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Synthesis of sugar conjugates: metal complexes and other derivatives Adam, Michael James 1980

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SYNTHESIS OF SUGAR CONJUGATES: METAL COMPLEXES AND OTHER DERIVATIVES by MICHAEL JAMES ADAM B . S c , The U n i v e r s i t y of B r i t i s h Columbia, 197 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Chemistry) We accept t h i s t h e s i s as conforming to the req u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA February 1980 iMichael James Adam, 1980 In presenting th i s thesis in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers ity of B r i t i s h Columbia, I agree that the Library shal l make i t f ree ly avai lable for reference and study. I further agree that permission for extensive copying of th i s thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It i s understood that copying or publ icat ion of th i s thesis for f inanc ia l gain shal l not be allowed without my written permission. Department of The Univers ity of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date MA/eC^/ /g/frQ ABSTRACT A number of conjugates of carbohydrates were prepared. Metal conjugates were synthesized i n two d i f f e r e n t ways. F i r s t l y , c h e l a t e c o o r d i n a t i o n complexes were synthesized by forming s a l i c y l a l d i m i n e l i g a n d s d erived from combinations of amino sugars [ m e t h y l - 3 , 4 , 6 - t r i -J3-acetyl-2-amino-2-deoxy-3-D-glucopyranoside, 1,3,4,6-tetra-0-acetyl-2-amino-2-deoxy-8-D-glucopyranose or 2-amino-2-deoxy-a,B-D-glucopyranose (glucosamine)] and e i t h e r s a l i c y l a l d e h y d e or 3-formyl-2-hydroxy-benzoic a c i d w i t h subsequent complexation of these to copper ( I I ) , cobal t ( I I ) , and z i n c ( I I ) . A number of p h y s i c a l techniques were used to c h a r a c t e r i z e these complexes i n c l u d i n g esr spectroscopy, v i s i b l e a bsorption spectroscopy, mass spectrometry, nmr spectroscopy and magnetic s u s c e p t i b i l i t y measure-ments. From the data provided by these techniques the copper-sugar complexes derived from s a l i c y l a l d e h y d e were found i n general to have the usual b i s - b i d e n t a t e s t r u c t u r e . The copper complex derived from the amino-glycoside and 3-formyl-2-hydroxy-benzoic a c i d was found to be b i n u c l e a r i n s t r u c t u r e c o n t a i n i n g two sugar moieties and two copper atoms. ' The second approach to forming metal sugar conjugates c o n s i s t e d of s y n t h e s i z i n g organometallic n-complexes: f e r r o c e n y l - s u g a r conju-gates. A v a r i e t y of organic- and water-soluble compounds were formed by r e a c t i o n of amino, h y d r o x y l , or t h i o sugar groups w i t h s u i t a b l y s u b s t i t u t e d ferrocene d e r i v a t i v e s . Thus organic s o l u b l e products were obtained from combinations of the sugars [ l , 3 , 4 , 6 - t e t r a - 0 - a c e t y l - 2 -amino-2-deoxy- 3-D-glucopyranose, l-thio-2,3,4,6-tetra-0_-acetyl-g-D-glucopyranose, 1,2,5,6-di-0_-isopropylidene-a-D-glucof uranose and 1,2,3,4-di-O-isopropylidene-a-D-galactopyranose] w i t h 1 - and 1,1'-ferrocenecarbonyl c h l o r i d e s , N,N-dimethylaminomethylferrocene methio-dide, (1-hydroxymethylferrocene)-p-toluenesulphonate and 2 , 4 - d i c h l o r o -6-(1-hydroxymethylferrocene)-s-triazine. Water s o l u b l e products were prepared by d e a c e t y l a t i o n of some of the above compounds and by conden-s a t i o n of ferrocene carboxaldehyde w i t h glucosamine to form the c o r r e s -ponding S c h i f f ' s base. Proton s p i n - l a t t i c e r e l a x a t i o n r a t e s were used to a s s i g n the s u b s t i t u t e d c y c l o p e n t a d i e n y l r i n g s and to determine the r e l a t i v e s p i n -ning r a t e s of the s u b s t i t u t e d and u n s u b s t i t u t e d c y c l o p e n t a d i e n y l r i n g s . The chemistry of cyanuric c h l o r i d e ( 2 , 4 , 6 - t r i c h l o r o - s - t r i a z i n e ) , as a general means of d e r i v a t i z i n g carbohydrates was a l s o i n v e s t i g a t e d . Thus, metals, hydrophobic a l k y l groups and n i t r o x i d e s p i n l a b e l s were attached i n v a r i o u s combinations to carbohydrates. A number of mono-saccharide d e r i v a t i v e s were formed i n c l u d i n g model g l y c o l i p i d s and a number of p o l y s a c c h a r i d e s , c e l l u l o s e , agarose, Sephadex, guar gum, xanthan gum and s t a r c h were s p i n l a b e l l e d using t h i s chemistry. For p o l y s a c c h a r i d e s , i n f o r m a t i o n such as extent of d e r i v a t i z a t i o n , evidence f o r a covalent bond, environment of the t r i a z i n e u n i t and the d i s t a n c e between t r i a z i n e u n i t s was obtained. This chemistry was a l s o extended to d e r i v a t i z e Bovine Serum Albumin, microporous g l a s s beads and aluminum oxide. i i i TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES v i LIST OF FIGURES v i i INTRODUCTION 1 Chapter I SYNTHESIS OF METAL CHELATE CONJUGATES OF AMINO SUGARS: SCHIFF'S BASE COMPLEXES 7 IA: I n t r o d u c t i o n 7 IB: Synthesis 10 ( i ) S c h i f f ' s Base Formation 10 ( i i ) S c h i f f ' s Base Metal Complexes 18 IC: P h y s i c a l Measurements 26 ( i ) V i s i b l e Absorption Studies 26 ( i i ) Esr Spectroscopy 31 ( i i i ) Magnetic Moments 40 ( i v ) Mass Spectrometry 41 • (v) Nmr Spectroscopy 44 ID: Other Chemistry 47 IE: Esr Spectroscopy of Copper ( I I ) 50 ( i ) General Esr 50 ( i i ) Copper ( I I ) Esr 51 IF: Summary and Conclusions 57 I I SYNTHESIS OF SUGAR-ORGANOMETALLIC CONJUGATES: FERROCENYL-MONOSACCHARIDE DERIVATIVES 63 IIA: I n t r o d u c t i o n 63 IIB: Synthesis 66 IIC: Proton Nuclear Magnetic Resonance Spectra . . 74 ( i ) Chemical S h i f t s and Coupling Constants 74 ( i i ) Proton Spin L a t t i c e R e l a x a t i o n Rates 86 IID: Proton S p i n - L a t t i c e R e l a x a t i o n 94 I I I CYANURIC CHLORIDE; A GENERAL REAGENT FOR THE CHEMICAL MODIFICATION OF CARBOHYDRATES ....... . 102 I I I A : I n t r o d u c t i o n 102 I I I B : Synthesis 109 ( i ) Nitrogen Nucleophiles 109 ( i i ) Oxygen and S u l f u r Nucleophiles . . . 115 ( i i i ) Summary 122 i v I I I C : Macromolecule and Surface M o d i f i c a t i o n . . . . 125 ( i ) P olysaccharides 125 (a) The General Reaction 125 (b) O p t i m i z a t i o n and Q u a n t i t a t i o n . . 129 (c) Evidence f o r a Covalent Bond . . . 138 (d) Distance Measurements 140 ( i i ) Bovine Serum Albumin 144 ( i i i ) Aluminum Oxide 144 ( i v ) C o n t r o l l e d Pore Glass 150 (v) Summary and Conclusions 154 HI D : Nmr Spectroscopy 157 H I E : Esr Spectroscopy of N i t r o x i d e s 162 IV SUMMARY 184 V EXPERIMENTAL 188 VA: E l e c t r o n Spin Resonance 188 VB: Nmr Measurements 190 VC: General Synthetic Procedures . . . 190 VD: Chapter I 191 ( i ) Sources of M a t e r i a l s 191 ( i i ) L i t e r a t u r e Preparations 191 ( i i i ) Synthesis 194 VE: Chapter I I 198 ( i ) Sources of M a t e r i a l s 198 ( i i ) L i t e r a t u r e Preparations 198 ( i i i ) Synthesis of Ferrocenyl-Sugar Conjugates 200 VF: Chapter I I I 207 ( i ) Sources of M a t e r i a l s 207 ( i i ) L i t e r a t u r e Preparations 208 ( i i i ) P o lysaccharide and Surface D e r i v a t i z a t i o n 209 ( i v ) Synthesis of s - T r i a z i n e Compounds . . . . 210 v LIST OF TABLES Table CHAPTER I 1-1. Esr parameters f o r copper ( I I ) complexes 32 1-2. Magnetic moments of copper complexes 41 CHAPTER I I I I - l . Chemical s h i f t s (ppm) and m u l t i p l e t s p l i t t i n g s (Hz) f o r the f e r r o c e n y l monosaccharide compounds . . . . 75 I I - 2 . Proton s p i n l a t t i c e r e l a x a t i o n r a t e s 90 CHAPTER I I I I I I - l . Data obtained from elemental a n a l y s i s and esr double i n t e g r a t i o n of l a b e l l e d polysaccharides . . . 135 I I I - 2 . Distances measurements determined f o r polysac-charides l a b e l l e d w i t h [22] 143 I I I - 3 . Extent of d e r i v a t i z a t i o n of AJI2O3 w i t h reagent [22] . 147 I I I - 4 . C o r r e l a t i o n times f o r n i t r o x i d e s i n chloroform s o l u t i o n 173 v i LIST OF FIGURES Figure CHAPTER I 1-1. 270 MHz ^H spectrum of compound [8] 14 T-2. 270 MHz 1E spectrum of compound [10] 15 1-3. 270 MHz *H spectrum of compound [11] 16 1-4. Decoupling experiment r e v e a l i n g hydrogen bonding i n compound [11] 19 1-5. V i s i b l e a bsorption s p e c t r a (CHC£3) comparing compounds [16] and [19] to p r e v i o u s l y known a l k y l complexes 27 1-6. Ambient temperature chloroform s o l u t i o n esr s p e c t r a of compounds [16] and [19] , n-butyl complex, i s o -p r o p y l complex, and t - b u t y l complex 34 1-7. 77K l i q u i d N2 frozen s o l u t i o n esr spectra of compounds [16] and [19], n - b u t y l complex, i s o -pr o p y l complex, t - b u t y l complex, and room temper-ature powder spectrum 35 1-8. Aqueous room temperature esr spectrum and the 77K fro z e n aqueous esr spectrum of compound [21] . . . 37 I - 9. The 77K frozen powder esr spectrum of the mono-nuclear copper complex impurity w i t h i n the b i n u c l e a r complex [23] 39 1-10. Sim u l a t i o n of the mass s p e c t r a l isotope p a t t e r n f o r a b i n u c l e a r copper complex 43 I - 11. Proton nmr spectrum (270 MHz) of the copper sugar complex [16] i n deu t e r i o c h l o r o f o r m 46 1-12. Proton nmr spectrum (270 MHz) of the z i n c complex [17] and the l i g a n d [8] 48 1-13. Spin s t a t e energy l e v e l diagram f o r a copper ( I I ) n u c l e i i n a magnetic f i e l d 53 1-14. Energy l e v e l diagram f o r two i n t e r a c t i n g copper ( I I ) n u c l e i i n a magnetic f i e l d 54 1-15. Schematic r e p r e s e n t a t i o n of the esr spectrum of copper ( I I ) 56 CHAPTER I I I I - l . Some t y p i c a l aromatic s u b s t i t u t i o n r e a c t i o n s of ferrocene 65 I I - 2 . Proton nmr s p e c t r a (270 MHz) of compound [7] i n deuteriobenzene 80 I I - 3. Proton nmr s p e c t r a (270 MHz) of compound [6] i n deuteriobenzene 81 v i i I I - 4 . Proton nmr spectrum (270 MHz) of compound [8] i n deuteriobenzene 82 I I - 5. Proton nmr s p e c t r a (270 MHz) of compound [23] i n deuterioacetone 84 I I - 6. P a r t i a l proton nmr spec t r a of compounds [6] and [7] i n deuteriobenzene 89 I I - 7 . Proton R i - v a l u e s f o r the sugar moiety of compound [6] 91 I I - 8. R o t a t i n g reference frame model 97 I I - 9. P l o t of magnetization vs t/Ti 98 CHAPTER I I I I I I - l . S t r u c t u r a l formulae of the polysaccharides s p i n l a b e l l e d v i a cyanuric c h l o r i d e 127 I I I - 2 . Ambient temperature aqueous esr spec t r a of s p i n l a b e l l e d polysaccharides 132 I I I - 3. Comparison of the aqueous esr spec t r a of l a b e l l e d Sephadex and Agarose w i t h t h e i r chloroform s p e c t r a 134 I I I - 4 . C o n t r o l esr experiments as evidence f o r covalent bonding of t r i a z i n e l a b e l to polysaccharides . . . . 141 I I I - 5. Powder spectrum showing the heights dj and d and the s p l i t t i n g 2A 142 I I I - 6. Ambient temperature aqueous esr spectrum of BSA l a b e l l e d w i t h reagent [22] 145 I I I - 7 ( i ) . Esr spectrum of l a b e l l e d alumina 146 I I I - 7 ( i i ) . Ambient temperature chloroform esr s p e c t r a of alumina l a b e l l e d w i t h [22] and alumina l a b e l l e d w i t h the non-reactive reagent [11] 149 I I I - 8 . Ambient temperature chloroform esr spec t r a of successive s p i n d i l u t i o n of n i t r o x i d e s on alumina 151 I I I - 9 . 77K frozen chloroform esr s p e c t r a of l a b e l l e d alumina before and a f t e r s p i n d i l u t i o n . . 152 111-10. Ambient temperature methanol esr spectrum of s i l i c a l a b e l l e d w i t h reagent [22]. 154 I I I - l l . Proton nmr spectrum (270 MHz) of compound [21] i n dueterioacetone 158 111-12. Proton nmr spectrum (270 MHz) of compound [32] . . . . 159 111-13. Proton nmr spectrum (270 MHz) of compound [36] before and a f t e r r e d u c t i o n 160 111-14. Energy l e v e l diagram f o r a n i t r o x i d e i n a magnetic f i e l d 165 111-15. D i r e c t i o n a l dependence of Zeeman and hyper f i n e i n t e r a c t i o n s 166 111-16. Esr spec t r a of ma g n e t i c a l l y d i l u t e d i - t - b u t y l n i t r o x i d e showing g and A a n i s o t r o p i e s 167 111-17. Esr s p e c t r a of [11] H0S£ i n an i n c r e a s i n g l y v i s c o u s s o l u t i o n 169 111-18. Esr spectrum of d i l u t e methanol s o l u t i o n s of the n i t r o x i d e [11] H0S£ and the n i t r o x i d e [36] 170 v i i i 111-19. E f f e c t s of the r a t e of exchange between A and B i n magnetic resonance spe c t r a 175 111-20. 77K i n f i n i t e d i l u t i o n chloroform esr s p e c t r a of the monoradical [22] and the b i r a d i c a l [25] 180 111-21. Ambient temperature chloroform esr s p e c t r a of the monoradical [11] and the b i r a d i c a l [25] 181 i x ACKNOWLEDGEMENT I am very g r a t e f u l to Dr. L. D. H a l l f o r h i s constant supply of encouragement, h e l p f u l d i s c u s s i o n s and moral support, which made the work described i n t h i s t h e s i s e x c i t i n g and rewarding. Although too numerous to name i n d i v i d u a l l y , I would l i k e to thank the graduate students, post d o c t o r a l f e l l o w s , and v i s i t i n g p r o f e s s o r s who were present i n t h i s lab during my Ph.D. work, f o r they, to a l a r g e degree, were my teachers. In p a r t i c u l a r , I would l i k e to thank Kim Wong, John Waterton, John A p l i n , Jeremy Saunders, and Mansur Y a l p a n i f o r many h e l p f u l d i s c u s s i o n s i n r e l a t i o n to t h i s t h e s i s work. I would a l s o to thank Drs. Geoffrey H e r r i n g and R. C. Thompson, along w i t h other members of the Chemistry Department, f o r many s t i m u l a t i n g d i s c u s -s i o n s and the N a t i o n a l Research C o u n c i l of Canada f o r f i n a n c i a l support. F i n a l l y , I am e s p e c i a l l y indebted to the l a t e P r o f e s s o r L. J . Muenster f o r the generous loan of equipment and f o r teaching me most of what I know about p r a c t i c a l s y n t h e t i c chemistry. x INTRODUCTION Carbohydrates are very important and abundant molecules i n nature as they play a v i t a l r o l e i n the biochemistry of v i r t u a l l y a l l l i v i n g t h i n g s . Both mono- and poly-saccharides are f i n d i n g an ever i n c r e a s i n g l y wide range of i n d u s t r i a l and biochemical a p p l i c a t i o n s ; areas of use 1 2 range from new pharmaceuticals and chromatography m a t e r i a l s through to 3 a d d i t i v e s f o r use i n the food i n d u s t r y and o i l recovery and the develop-ment of renewable sources of energy . These and other areas would c l e a r l y be advanced both by general techniques f o r changing the p h y s i c a l c h a r a c t e r i s t i c s of carbohydrates and by the development of methods f o r studying the s t r u c t u r e and f u n c t i o n of " n a t u r a l " and "modified" sacchar-ides . A b a s i c p r e r e q u i s i t e of any of these s t u d i e s , i s the development of a general methodology i n which carbohydrates can be chemically modi-f i e d , p r e f e r a b l y i n a s e l e c t i v e f a s h i o n . The main body of work i n t h i s t h e s i s c o n s i s t s of the development of chemical procedures whereby a v a r i e t y of chemical l i g a n d s can be attached to both mono- and po l y -saccharide f a m i l i e s of carbohydrates. At t h i s point i t i s convenient to d i s c u s s the t h e s i s as i f i t c o n s i s t e d of e s s e n t i a l l y two p a r t s , the f i r s t i n v o l v e s the development of s p e c i f i c chemistry f o r the attachment of metals to carbohydrates and the second describes the development of a v e r s a t i l e chemistry whereby many d i f f e r e n t types of chemical groups can be attached. 1 2 Due to a long standing i n t e r e s t by our lab i n metal-sugar conju-gates (the reasons f o r which w i l l be given l a t e r ) , the synthesis of s p e c i f i c metal sugar compounds was i n i t i a l l y undertaken. Metal complexes of S c h i f f ' s bases have occupied a c e n t r a l r o l e i n the development of c o o r d i n a t i o n chemistry"* and have been known f o r over one hundred years d a t i n g as f a r back as 1840. Since amino sugars are important b i o l o g i c a l molecules and are c o n s t i t u e n t s of a l a r g e number of a n t i b i o t i c s , i t might be expected that the combined presence of sugar and metal moieties would give these compounds i n t e r e s t i n g b i o l o g i c a l p r o p e r t i e s . Therefore my f i r s t experiments i n t h i s area were designed to form S c h i f f ' s base l i g a n d s by combining amino sugars w i t h s u i t a b l e aldehydes or ketones, and subsequently complexing these w i t h metals. Although the chemistry of ferrocene and other aromatic ^-complexes i s not as o l d as that of S c h i f f ' s - b a s e complexes i t s t i l l goes back as f a r as 1948^ and i s now very w e l l documented. These subtances c o n s t i -t u t e a c e n t r a l r o l e i n organometallic chemistry and t h e i r a p p l i c a t i o n s i n chemistry, biochemistry, and i n d u s t r y have grown d r a m a t i c a l l y over the l a s t ten years . A c c o r d i n g l y , the second round of experiments w i t h regard to sugar metal conjugates i n v o l v e d the formation of organometallic carbohydrate complexes, s p e c i f i c a l l y f e r r o c e n y l - s u g a r conjugates. The p r e p a r a t i o n of these substances has i n v o l v e d a v a r i e t y of monosacchar-ides i n c l u d i n g amino- and t h i o - s u g a r s . The methods used f o r the d i r e c t conjugation of metals to carbo-hydrates v i a these two approaches were, however, thought to be somewhat l i m i t e d i n scope and i t seemed l o g i c a l to t r y and develop chemistry which would enable one to l i n k a l a r g e r v a r i e t y of organic and i n o r g a n i c m a t e r i a l s to carbohydrates. To achieve t h i s d i v e r s i t y the chemistry that was i n v e s t i g a t e d i n v o l v e d the v e r s a t i l e c o u p l i n g reagent cyanuric c h l o r i d e ( 2 , 4 , 6 - t r i c h l o r o - s - t r i a z i n e ) . This substance has been known sinc e 1827 and i t s chemistry i s very w e l l understood. I t has been wid e l y used i n i n d u s t r y i n areas ranging from the d y e s t u f f s i n d u s t r y , where i t i s used f o r making r e a c t i v e dyes f o r c e l l u l o s e f i b e r s ^ to i t s 9 11 use i n the manufacturing of rubber and p l a s t i c s ', and i n the preparation 9 12 of medicinals such as a n t i b a c t e r i a l s and a n t i - c a n c e r drugs ' Cyanuric c h l o r i d e 1 i s a h e t e r o c y c l i c compound c o n t a i n i n g three c h l o r i n e s u b s t i t u e n t s which can be d i s p l a c e d by many d i f f e r e n t nucleo-p h i l e s under a v a r i e t y of c o n d i t i o n s . What makes t h i s reagent so CI C I ^ N ^ C I [11 a t t r a c t i v e i s that u n l i k e most other b r i d g i n g groups or coupling reagents, which are u s u a l l y b i f u n c t i o n a l , i t possesses three p o i n t s of attachment to which other m o i e t i e s can be bonded, and hence i t can be termed a " t r i v a l e n t l o c u s " . Using t h i s reagent i t has been p o s s i b l e to form v a r i o u s combinations of metal compounds, hydrophobic and h y d r o p h i l i c e n t i t i e s , and s p i n l a b e l n i t r o x i d e f r e e r a d i c a l r e p o r t e r groups. An example of one such u s e f u l combination i s i l l u s t r a t e d by the synthesis of model g l y c o l i p i d s which can be made w i t h or without a s p i n l a b e l r e p o r t e r group attached. Such molecules may have uses i n probing the f u n c t i o n s of "model" and " r e a l " membranes and i n studying phenomena 4 13 such as c e l l - c e l l i n t e r a c t i o n s I t was thought important to demonstrate that the above chemistry can a l s o be a p p l i e d outside the realm of carbohydrate chemistry, and examples w i l l i n c l u d e the s p i n l a b e l l i n g of Bovine Serum Albumin (BSA), and the use of cyanuric c h l o r i d e i n the d e r i v a t i z a t i o n of other s o l i d support matrices such as aluminum oxide and g l a s s . Along w i t h the s y n t h e t i c methodology, s u i t a b l e s p e ctroscopic t o o l s had to be developed i n order to prove the s t r u c t u r e s of the various products, some of which had r a t h e r unusual compositions. Both esr and nmr spectroscopy were used e x t e n s i v e l y throughout the course of the work. In chapter I I I , n i t r o x i d e f r e e r a d i c a l esr w i l l be discussed w i t h p a r t i c u -l a r emphasis on i t s use i n r e l a y i n g (a) i n f o r m a t i o n concerning the 14 15 environment of the l a b e l , (b) i n measuring distances , and (c) i n 16 determining the extent of chemical d e r i v a t i z a t i o n . Using these tech-niques i t w i l l be shown how esr spectroscopy can be used to "debug" the chemistry of cyanuric c h l o r i d e as i t i s used i n the d e r i v a t i z a t i o n of s o l i d support matrices. I t i s w e l l known that esr i s a l s o a good spectroscopic t o o l f o r the s t r u c t u r e determination of c e r t a i n t r a n s i t i o n metal complexes"^, and i n chapter I i t w i l l be discussed i n the context of determining s t r u c t u r a l i n f o r m a t i o n about the copper sugar complexes synthesized t h e r e i n . Nmr spectroscopy w i l l be a l l u d e d to throughout the t e x t as a general method f o r the s t r u c t u r a l determination of organic compounds. However, a more s p e c i a l i z e d nmr experiment w i l l be explained i n chapter I I , where proton s p i n l a t t i c e r e l a x a t i o n w i l l be demonstrated 18 as a technique f o r a s s i g n i n g proton resonances and f o r determining motional c o r r e l a t i o n times f o r some of the f e r r o c e n y l - s u g a r compounds synthesized. 5 References 1. H. H. Baer and J . L. Strominger, The Amino Sugars, The Chemistry and Biology of Compounds Containing Amino Sugars, Academic P r e s s , New York, 1A, 1969; R. L. W h i s t l e r , I n d u s t r i a l Gums, Pol y s a c - charides and Their D e r i v a t i v e s , Academic P r e s s , New York, 1973. 2. H. F. Hixson, J r . and E. P. Goldberg, Polymer G r a f t s i n Biochemistry, Dekker, New York, 1976. 3. R. L. W h i s t l e r , I n d u s t r i a l Gums, Academic P r e s s , New York, 1973; C. T. Githens and J . W. Burnham, Society of Petroleum Engineers J o u r n a l , _T7_, 5(1977); R. T i n e r , Southwestern Petroleum Short Course, Proceedings, 2j$, 1976; B. Sand i f o r d , Energy Communications, _4, 53(1978); H. E. G i l l i l a n d , Energy Communications, 4_, 83(1978). 4. V. K o l l o n i t s c h , Sucrose Chemicals, The I n t e r n a t i o n a l Sugar Research Foundation Inc., W. S. Cowell L t d . , Great B r i t a i n , 1970. 5. R. H. Holm, G. W. E v e r e t t , J r . , and A. Chakravorty, Progr. Inorg. Chem. , ]_, 83(1966). 6. R. W. Je a n l o z , The Amino Sugars, Academic P r e s s , New York, Ik, 1969. 7. G. Wil k i n s o n and F. A. Cotton, Progr. Inorg. Chem., 1, 1(1959). 8. B. W. Rockett and G. Marr, J . Organomet. Chem., 167, 53(1979). 9. E. M. Smolin and L. Rapoport, The Chemistry of H e t e r o c y c l i c Compounds, 13, 1959. 10. R. L. M. A l l e n , Color Chemistry, Appleton-Century-Crofts, New York, 1971. 11. W. R. S t i n e , Chemistry f o r the Consumer, A l l y n and Bacon, Inc., Toronto, 1978. 12. A. F u r s t , Chemistry of C h e l a t i o n i n Cancer, Charles C. Thomas (pub-l i s h e r ) , S p r i n g f i e l d I l l i n o i s , 1963. 13. J . C. Waterton and A. van der E s t , manuscript i n p r e p a r a t i o n . 14. L. J . B e r l i n e r , Spin L a b e l l i n g , Theory and A p p l i c a t i o n , Academic Pr e s s , New York, 1976. 15. A. L. Kokorin, K. I . Zamarayer, G. L. Grigoryan, V. P. Ivanov and E. G. Rosantzev, B i o f i z i k a 17_, 34(39 i n t r a n s l . ) (1972). 16. J . D. A p l i n , Ph.D. t h e s i s , U n i v e r s i t y of B r i t i s h Columbia, 1979. 17. B. J . Hathaway and D. E. B i l l i n g s , Coordination Chem. Reviews, _5, 143(1970); H. Yokoi, B u l l . Chem. Soc. Japan, 47, 3037(1974). 6 18 L. D. H a l l , Chem. Soc. Rev., 4, 401(1975); L. D. H a l l , Chemistry i n Canada, 28, 19(1976); J . K. M. Saunders, Annual Reports Chem. Soc. B, 75, 3(1979). CHAPTER I SYNTHESIS OF METAL CHELATE CONJUGATES OF AMINO SUGARS: SCHIFF'S BASE COMPLEXES IA: I n t r o d u c t i o n The f a c t that sugars form complexes w i t h metals has been known f o r 1 2 about 100 years ' . However, the f i e l d of sugar-metal complexes i s s t i l l l a r g e l y unexplored. The chemical l i t e r a t u r e r e c o r d s , s i n c e 1973, at l e a s t 70 c r y s t a l l i n e complexes which cont a i n a sugar and an i n o r g a n i c s a l t . These complexes are, however, mainly r e s t r i c t e d to group IA and group IIA metals, and the s t r u c t u r e s of these metal sugar "adducts" i s , f o r the most p a r t , unknown. These complexes are, though, very important i n the b i o l o g y of a l l l i v i n g things s i n c e i t has been shown that sugars are an important f a c t o r i n the a c t i v e c e l l u l a r t r a n s p o r t of calcium and other group IIA metals . More r e c e n t l y , i t has been shown that c y c l i t o l s and sugars which c o n t a i n an a x i a l - e q u a t o r i a l - a x i a l sequence of three hydroxyl groups i n a six-membered r i n g , or a c i s - c i s sequence i n a f i v e -membered r i n g , form 1:1 complexes w i t h metal c a t i o n s i n a h y d r o x y l i e OH OH OH 7 2 3 solvent ' . Complex formation has a l s o been found to cause a change i n the conformational e q u i l i b r i u m and the anomeric e q u i l i b r i u m of various 2 sugars . Complex formation a l s o makes p o s s i b l e the separation of some sugars by e l e c t r o p h o r e s i s and ion-exchange chromatography. L i t e r a t u r e on the complexation of sugars w i t h t r a n s i t i o n metals and metals other than those i n group I and IIA i s i n comparison much more recent and l e s s abundant. Some of these s t u d i e s have included i r o n 4 sugar complexes f o r use as hema'tinic agents ( i r o n d e f i c i e n c y drugs) , n i c k e l , c o b a l t , i r o n , manganese complexes of glucosaminic a c i d ^ , copper, l e a d , z i n c , n i c k e l and cadmium complexes of D-glucosamines f o r the study of a n t i b i o t i c f u n c t i o n s ^ , l i t h i u m aluminum hydride sugar complexes as asymmetric reducing agents^, i n t e r a c t i o n s of metals w i t h amino-sugar c o n t a i n i n g a n t i b i o t i c s , copper caped c y c l o d e x t r i n s f o r mimicing enzyme 9 bi n d i n g s i t e s , copper cobalt and n i c k e l complexes of nitr o g e n - h e t e r o -c y c l i c monosaccharide d e r i v a t i v e s as analogues f o r nucleoside and nu c l e o t i d e metal complexes 1^, rhodium diphenylphosphine sugar complexes as asymmetric hydrogenation catalyst''""'", rhodium complexes of d i p h e n y l -12 phosphine d e r i v a t i z e d c e l l u l o s e , and copper complex formation w i t h 13 c e l l u l o s e s . In our own l a b o r a t o r y , d i r e c t carbon-metal bonded complexes of mercury 1^, t h a l l i u m 1 " ' , t i n 1 ^ , and v i t a m i n B^g, cyclopenta-d i e n y l i r o n , tungsten, and molybdenum t r i a r b o n y l 1 ^ sugar d e r i v a t i v e s have a l s o been prepared. I t i s c l e a r then that the a b i l i t y of sugars to sequester metals i s of current i n t e r e s t f o r a wide v a r i e t y of reasons. Some of the more important a p p l i c a t i o n s of t h i s chemistry i n c l u d e s the p o s s i b l e develop-ing ment of novel c l a s s e s of metal based a f f i n i t y chromatography m a t e r i a l s , c h i r a l homogeneous c a t a l y s t s 1 1 , metal c h e l a t o r s f o r c l i n i c a l use", and 4 of models f o r b i o l o g i c a l l y important chelates"*"^. Further i n t e r e s t s 8 19 i n c l u d e nmr s t u d i e s of the i n t e r a c t i o n of many metals w i t h sugars ' These and many other s t u d i e s would be advanced by the continued develop-ment of general methods whereby metals could be r e a d i l y attached to sugars. Metal complexes of S c h i f f ' s bases have occupied a c e n t r a l r o l e i n 20 the development of c o o r d i n a t i o n chemistry and have been known f o r over one hundred years, d a t i n g as f a r back as 1840. A tremendous v a r i e t y of s t a b l e chemical species have been synthesized c o n t a i n i n g both t r a n s i t i o n and non t r a n s i t i o n metals. The most important groups of S c h i f f ' s base complexes are the complexes of s a l i c y l a l d i m i n e s and 8-ketoamines, and c l o s e l y r e l a t e d systems. S c h i f f ' s bases are those compounds c o n t a i n i n g the azomethine group (-RC=N-) and are u s u a l l y formed by the condensation of a primary amine wi t h an a c t i v e carbonyl compound. Bases which are e f f e c t i v e as c o o r d i n -a t i n g l i g a n d s bear a f u n c t i o n a l group, u s u a l l y -OH, s u f f i c i e n t l y near the s i t e of condensation that a f i v e - or six-membered chelate r i n g can be formed upon r e a c t i o n w i t h a metal i o n . Because of the great f l e x i b i l i t y of S c h i f f ' s base formation, many lig a n d s of d i v e r s e s t r u c t u r a l type can be and have been synthesized. Amino sugars form S c h i f f ' s bases r e a d i l y w i t h s a l i c y l a l d e h y d e and 21 22 other aromatic aldehydes and have been known sin c e 1922 when I r v i n e and E a r l f i r s t showed that s p a r i n g l y water s o l u b l e S c h i f f ' s bases of glucosamine could be formed w i t h s a l i c y l a l d e h y d e . These S c h i f f ' s base d e r i v a t i v e s were found to provide a good means f o r i s o l a t i n g amino sugars 23 from hydrolyzed polysaccharides and p r o t e i n s In t h i s chapter the formation of metal complexes of amino sugar-10 S c h i f f ' s base d e r i v a t i v e s w i l l be discussed. F i r s t the s y n t h e s i s of s a l i c y l a l d e h y d e and 3-formyl-2-hydroxy-benzoic a c i d S c h i f f ' s base l i g a n d s derived from both alkylamine and glucosamine d e r i v a t i v e s along w i t h t h e i r metal complexes w i l l be described. Then the p h y s i c a l techniques of v i s i b l e absorption spectroscopy, e s r , and nmr spectroscopy, mass spectrom-e t r y and magnetic moments w i l l be examined as t o o l s f o r the s t r u c t u r e determination of the complexes synthesized. F o l l o w i n g t h i s , a s e c t i o n on "other chemistry" w i l l be presented which describes r e a c t i o n s which d i d not y i e l d the d e s i r e d product but which do provide some i n t e r e s t i n g r e s u l t s . Nmr data on the v a r i o u s diamagnetic l i g a n d s w i l l be presented throughout the chapter as a r o u t i n e s t r u c t u r a l t o o l . F i n a l l y , a s e c t i o n d e s c r i b i n g some of the b a s i c features of esr spectroscopy as i t p e r t a i n s to s t u d i e s of copper ( I I ) complexes w i l l be presented. When esr data are given e a r l i e r i n s e c t i o n I C ( i i ) the reader not f a m i l i a r w i t h t h i s technique w i l l be r e f e r r e d ahead to t h i s l a s t s e c t i o n . IB: Synthesis ( i ) S c h i f f ' s Base Formation As mentioned i n the i n t r o d u c t i o n , S c h i f f ' s bases are those com-pounds c o n t a i n i n g the azomethine group which are u s u a l l y formed by the condensation of a primary amine w i t h an a c t i v e carbonyl compound. The a c t i v e carbonyl compounds considered here are 2-hydroxy-benzaldehyde ( s a l i c y l d e h y d e ) [1] and 3-formyl-2-hydroxy-benzoic a c i d (3-aldehydo-s a l i c y l i c a c i d ) [ 2 ] . A t y p i c a l S c h i f f ' s base formation, w i t h c y c l o h e x y l -amine and s a l i c y l a l d e h y d e i s shown below ([3] and [ 4 ] ) . Just as alkylamines r e a d i l y form S c h i f f ' s bases, so do amino sugars. S a l i c y l a l d i m i n e compounds ( S c h i f f ' s bases formed from s a l i c y l a l d e h y d e or 11 [4] substituted salicylaldehyde compounds) from glucosamine (2-amino-2-deoxy-22 a,B-D-glucopyranose) were f i r s t synthesized by Irvine and Earl in 1922 The reaction between glucosamine hydrochloride [5] and salicylaldehyde, as shown below, is very easily accomplished with the water insoluble [6] product [6] being obtained in a high yield. As mentioned in the intro-duction, this reaction was found to be useful for the isolation of amino sugars, such as glucosamine, from hydrolysis extracts of proteins and 12 polysaccharides. As well as "free" glucosamine, "blocked" glucosamine derivatives such as methyl 3,4,6-tri-0-acetyl-2-amino-2-deoxy-B-D-glucopyranoside 24 hydrobromide [7] and l,3,4,6-tetra-0_-acetyl-2-amino-2-deoxy-B-D-gluco-25 pyranose [9] also condense with salicylaldehyde to form the Schiff's bases [8] and [10] respectively, as shown below. [10] Schiff's bases formed from 3-aldehydo s a l i c i c acid [2] have, in com-parison to other salicylaldimines, received very l i t t l e attention. To my knowledge, Schiff's base derivatives formed between amino sugars and 3-aldehydo-salicylic acid have not before been reported. The formation of the Schiff's base between the blocked glucosamine sugar [7] and this aldehyde is also very easily accomplished giving compound [11] in a high 13 OAc OAc 0 = C H M e O H H,0 AcO AcO N = C H NH 3 Br [7] [2] [11] y i e l d . The sugar S c h i f f ' s base compounds [6], [8], [10] and [11] are c r y s t a l l i n e stable compounds as are t h e i r a l k y l counterparts and are bright yellow i n c o l o r . These compounds are, however, a c i d - and base-l a b i l e due to t h e i r azomethine linkage. Because of t h i s , aromatic aldehydes can be used conveniently as amine blocking groups for amino sugars. Sugar S c h i f f ' s bases are, fortunately, stable to cold anhydrous 25 a c y l a t i n g reagents; thus the amino sugar [9] was prepared by using anisaldehyde (p-methoxy-benzaldehyde) [12] as an amino blocking group with subsequent a c e t y l a t i o n and S c h i f f ' s base and cleavage as shown below. Metal complexation using ligands [6], [8], [10] and [11] w i l l be the main f o c a l point of t h i s chapter. In order for metal complexation to occur with these compounds, the hydroxyl function on the aromatic r i n g must be brought into close proximity with the nitrogen atom of the sugar group. Therefore, the aromatic hydroxyl group should preferably be strongly hydrogen bonded to the nitrogen atom of the sugar r i n g as shown below for [10]. Evidence for t h i s hydrogen bonding comes from the 1H nmr spectra of compounds [8], [10] and [11] which are shown i n Figures 1-1, 2 and 3 r e s p e c t i v e l y . The hydroxyl protons for [8], [10] and [11] Figure 1-1. 270 MHz *H spectrum of compound [8]. H 3 H , OH 12 H O N 8 j L L H J Figure 1-2. 270 MHz *H spectrum of compound [10] Figure 1-3. 270 MHz *H spectrum of compound [11]. 17 MO] are found at 12.A ppm, 12 ppm and 14 ppm respectively and such very low f i e l d chemical shifts are characteristic of hydrogen bonded protons. Compound [11] has a very interesting spectrum and additional information for the existence of a strong hydrogen bond in this molecule can be seen. In Figure 1-3 the imine proton and the H 2 sugar ring proton, are broader and more poorly resolved than normally expected. This i s a result of partial vicinal spin-spin coupling of these protons to the hydrogen bonded hydroxyl proton. By irradiating the hydroxyl 18 resonance at 14 ppm, the r e s u l t a n t sharpening of the imine and H2 pro-tons (Figure 1-4) c l e a r l y prove that coupling e x i s t s and hence proves the existence of hydrogen bonding. The reason the imine and H 2 reson-ances are only broadened r a t h e r than s p l i t i n t o a greater m u l t i p l i c i t y can be explained by assuming exchange of the hydroxyl proton. I f the exchange r a t e between the hydroxyl proton and the a c i d f u n c t i o n w i t h i n the molecule or between neighbouring molecules were very r a p i d , then the hydroxyl proton would be e f f e c t i v e l y "decoupled" from the imine and H 2 protons; and the hydroxyl and a c i d proton resonances would c o l l a p s e to one sharp l i n e . I f the exchange r a t e i s slow or non e x i s t e n t the imine and H 2 proton resonances would be s p l i t by coupling to the hydroxyl proton and would a l s o have narrow l i n e widths; and the hydroxyl and a c i d resonances would appear as two i n d i v i d u a l sharp l i n e s . Therefore, apparently exchange occurs at an intermediate r a t e thereby broadening the resonances of the imine and H 2 resonances as w e l l as the hydroxyl and a c i d resonances. In compounds [8] and [10] t h i s broadening e f f e c t i s not observed, e i t h e r because hydrogen bonding of the hydroxyl proton to the n i t r o g e n i s not strong enough f o r c o u p l i n g to be observed, or because the hydroxyl proton i s exchanging so r a p i d l y w i t h the hydroxyl group of another molecule that i t i s e f f e c t i v e l y decoupled from the imine and H 2 protons. ( i i ) S c h i f f ' s Base Metal Complexes As p r e v i o u s l y noted, of a l l S c h i f f ' s bases, those derived from 20 s a l i c y l a l d i m i n e s have been by f a r the most thoroughly stu d i e d . The p a r t i c u l a r advantage of the b a s i c s a l i c y l a l d i m i n e l i g a n d system i s the considerable f l e x i b i l i t y of the s y n t h e t i c procedure. As a r e s u l t , a wide v a r i e t y of complexes have been formed and by s t r u c t u r a l v a r i a t i o n s 19 HC=N 8 3.5 ppm Figure 1-4. Decoupling experiment r e v e a l i n g hydrogen bonding i n compound [11]. The spectrum i n (A) shows the sharpening of the imine and H2 protons upon i r r a d i a t i o n of the aromatic hydroxyl proton. Spectrum (B) i s the non-decoupled spectrum. of the l i g a n d systematic changes i n p r o p e r t i e s have been examined. S a l i c y l a l d i m i n e complexes are g e n e r a l l y r e a d i l y prepared and e a s i l y p u r i f i e d by r e c r y s t a l l i z a t i o n . B a s i c a l l y two s y n t h e t i c procedures have been employed. 1. Reaction of the metal i o n and S c h i f f base i n a homogeneous a l c o h o l , or aqueous a l c o h o l , s o l u t i o n w i t h the p o s s i b l e a d d i t i o n of a base such as acetate or hydroxide. 2. Reaction of a primary amine w i t h the preformed s a l i c y l a l d e h y d e - m e t a l complex. This preformed complex i s heated under r e f l u x w i t h the amine i n a solvent such as e t h a n o l , or chloroform f o r a p e r i o d of 1 h or l e s s and the crude product i s obtained by c o o l i n g and/or volume r e d u c t i o n . This r e a c t i o n may be represented as f o l l o w s . * *adapted from reference (20). By removing " e l e c t r o n d e n s i t y , " the coordinated metal i o n enhances the p o l a r i z a t i o n of the carbonyl group, thereby promoting n u c l e o p h i l i c a t t a c k by the amine-nitrogen. Although the l a t t e r s y n t h e t i c method has been used by other workers more oft e n than the f i r s t , i t was found i n t h i s study that method (1) was more a p p l i c a b l e f o r the formation of sugar complexes. The most s i g n i f i c a n t complexes of the s a l i c y l a l d i m i n e s are of the s t r u c t u r a l types (A) and (B) where R, X, and B are g e n e r a l i z e d n i t r o g e n , r i n g , and b r i d g i n g group s u b s t i t u e n t s , r e s p e c t i v e l y . The complexes discussed i n t h i s t h e s i s are r e s t r i c t e d to the s t r u c t u r a l type (A) i n which X i s e i t h e r a hydrogen or c a r b o x y l i c a c i d s u b s t i t u e n t . Instead of using the cumbersome systematic nomenclature the abbreviated form M(R-sal) w i l l be used, n A wide v a r i e t y of metals and R groups can be incorporated i n t o complexes of the s t r u c t u r a l type (A). T y p i c a l l y R can be n-propyl, n-decyl, t - b u t y l or c y c l o h e x y l , and M, Cu, N i , Co, Zn, Mn or Cr. The c y c l o h e x y l moiety i s the most s t r u c t u r a l l y s i m i l a r a l k y l group to the 20 pyranose sugar r i n g . I t i s known that when n = 2, the bis-complex has the oxygen and n i t r o g e n donor atoms arranged as shown i n the struc-ture of the c y c l o h e x y l complex [15]. 22 [15] The c y c l o h e x y l s a l i c y l a l d i m i n e l i g a n d can complex w i t h s e v e r a l d i v a l e n t metals to form many complexes of the b i s - s t r u c t u r e [15]. The analogous sugar s a l i c y l a l d i m i n e d e r i v a t i v e s [8] and [10] were ther e f o r e expected to form complexes e q u a l l y as w e l l (complexation using ligands [6] and [11] w i l l be discussed s e p a r a t e l y l a t e r ) . Indeed f o r copper ( I I ) t h i s was the case. When a methanol s o l u t i o n of c u p r i c acetate was added to a methanol s o l u t i o n of [8] at room temperature, the copper sugar complex [16] p r e c i p i t a t e d immediately as a brown s o l i d i n 90% y i e l d . * . -OAc J—°vOMe A c o ^ n OH / N = C H OAc [16] M=Cu [17] M = Zn [18] M = Co E M [Sug I-sal Ac 0-*For convenience, from now on, the sugar u n i t shown i n [16], [17] and [18] w i l l be abbreviated as Sug I . In complexes i n c o r p o r a t i n g the sugar u n i t s derived from compounds [9] and [ 5 ] , the sugar moiety w i l l be abbreviated to Sug I I and I I I r e s p e c t i v e l y . The i r spectrum showed a s h i f t i n the -C=N- absorption to lower frequency compared w i t h the l i g a n d ( [ 8 ] , 1630 cm "*"; [16], 1590 cm ^) i n d i c a t i n g 26 the c o o r d i n a t i o n of the n i t r o g e n atom to the copper i o n Both z i n c and cobaltous acetate a l s o complexed w i t h the l i g a n d [8] i n methanol to form the ye l l o w and green bis-complexes [17] and [18] r e s p e c t i v e l y . The y i e l d s f o r these complexes were, however, lower than the copper analogue, being 41% and 51% r e s p e c t i v e l y . Although s t a b i l i t y constants were not determined f o r these complexes, some evidence suggests that they are not as s t a b l e as t h e i r a l k y l counter-p a r t s . Thus the sugar complexes decomposed back to t h e i r parent l i g a n d s when run on s i l i c a g e l t h i n l a y e r chromatography ( t i c ) , whereas a l k y l complexes such as [15] are s t a b l e on s i l i c a g e l . A l s o , the z i n c and cob a l t complexes [17] and [18] decomposed when d i s s o l v e d i n hot methanol, and had to be r e c r y s t a l l i z e d under c o l d c o n d i t i o n s (see Experimental). The copper complex was, however, s t a b l e to t h i s treatment. Thus i t seems that the z i n c and coba l t complexes are l e s s s t a b l e than the copper complex. The sugar s a l i c y l a l d i m i n e l i g a n d [10] al s o forms a complex [19] Cu (Sug I I - s a l ) 2 w i t h copper; t h i s was obtained as shiny o l i v e green c r y s t a l s i n 90% y i e l d when a hot ethanol s o l u t i o n of c u p r i c acetate and l i g a n d [10] were mixed and allowed to c o o l . This complex could be r e c r y s t a l l i z e d from hot ethanol and decomposed only p a r t i a l l y on s i l i c a g e l t i c . However, attempted complexation of [10] w i t h z i n c and c o b a l -tous acetate f a i l e d to y i e l d any product. The decomposition mixture of the cobal t complex [18] i n hot meth-anol and the r e a c t i o n mixture between [10] and cobal t a c e t a t e , were both orange i n c o l o r . This same c o l o r was observed f o r a s o l u t i o n of the 24 s a l i c y l a l d e h y d e c o b a l t complex [20]. I t t h e r e f o r e seems l i k e l y that the azomethine bond i s being cleaved i n both these cases r e s u l t i n g i n the formation of the more s t a b l e s a l i c y l a l d e h y d e complex [20]. Probably the most s i g n i f i c a n t complex prepared from these sugar s a l i c y l a l d i m i n e s , was the copper complex of the f r e e sugar s a l i c y l a l -dimine [6]. This l i g a n d formed a copper complex [21] e q u a l l y as w e l l as the other l i g a n d s using the same r e a c t i o n c o n d i t i o n s . This copper-sugar complex i s h i g h l y water-soluble and, as mentioned e a r l i e r , the combined presence of a n a t u r a l l y o c c u r r i n g carbohydrate and metal i o n might have u s e f u l a p p l i c a t i o n s i n the pharmaceutical f i e l d . The s t r u c -t u r e of t h i s complex i s , however, l e s s c l e a r cut than the complexes d i s -cussed thus f a r , and the l i m i t e d evidence which has been obtained w i l l be discussed l a t e r . The p o s s i b l e s t r u c t u r e of t h i s complex based on data from a combination of elemental a n a l y s i s , esr and mass spectrometry i s shown below. [20] OH HO [21] In c o n t r a s t to the s a l i c y l a l d e h y d e derived s a l i c y l a l d i m i n e S c h i f f ' s base complexes, those formed from the 3 - a l d e h y d o - s a l i c y l i c a c i d S c h i f f ' s bases have not r e c e i v e d as much a t t e n t i o n . As discussed i n I B ( i ) these l i g a n d s can be r e a d i l y prepared i n high y i e l d i n the same way as the p r e v i o u s l y described s a l i c y l a l d i m i n e s . The n i t r o g e n s u b s t i t -uent can be q u i t e v a r i e d and a number of metals, mostly f i r s t row t r a n s i -27 t i o n , have been incorporated . The s t r u c t u r e proposed f o r these com-plex e s , where the n i t r o g e n s u b s t i t u e n t R i s non b r i d g i n g , i s shown below. H,0-= M(R-sa l icy l ic] As f o r the other s a l i c y l a l d i m i n e complexes, these complexes can be prepared simply by mixing the l i g a n d and metal s a l t , u s u a l l y the metal ace t a t e , i n an a l c o h o l s o l u t i o n , and then f i l t e r i n g the p r e c i p i t a t e d product from the r e a c t i o n mixture. The r e a c t i o n of the sugar l i g a n d [11] and c u p r i c acetate i n ethanol was accomplished i n j u s t t h i s way, r e s u l t i n g i n a l e a f - g r e e n product. As w i l l be discussed i n more d e t a i l l a t e r , mass spectrometry and esr spectroscopy s t r o n g l y suggest that the product has a b i n u c l e a r copper s t r u c t u r e as shown below, w i t h a s m a l l percentage of a mononuclear copper complex i m p u r i t y . This mononuclear complex a l s o contains the sugar moiety, as shown by esr spectroscopy [ I C ( i i ) ] and may w e l l be of the same s t r u c t u r e as that proposed i n [22]. Various p h y s i c a l methods can be used to determine the s t r u c t u r e s of 26 [23] these complexes and i n the next s e c t i o n the use of a v a r i e t y of these w i l l be described. IC: P h y s i c a l Measurements ( i ) V i s i b l e Absorption Studies In t h i s s e c t i o n , the use of v i s i b l e a bsorption spectroscopy f o r determining the c o o r d i n a t i o n geometry of the sugar-copper complexes Cu (Sug I - s a l ) 2 [16] and Cu (Sug I I - s a l ) 2 [19] and the cobal t complex Co (Sug I - s a l ) 2 [18] w i l l be examined. The data f o r [16] and [19] w i l l be compared to data obtained f o r a s e r i e s of Cu ( R - s a l ) 2 complexes of known geometry. Although the v i s i b l e absorption spectra of the Cu ( R - s a l ) 2 com-28 ' ple x e s , where R = n-Bu, i - P r , and t-Bu, have been reported elsewhere they have been measured here f o r comparison purposes, along w i t h the sugar complexes, i n order to obt a i n systematic i n f o r m a t i o n about t h e i r c o o r d i n a t i o n geometry; a l l of the observed sp e c t r a are shown i n Figure 1-5. Figure 1-5. V i s i b l e absorption spectra (CHC£ 3), comparing compounds [16] and [19] to p r e v i o u s l y known a l k y l complexes. 28 The c r y s t a l s t r u c t u r e of the complex Cu ( R - s a l ) 2 , where R = n-Bu, 30 shows that t h i s complex adopts a "normal" planar c o n f i g u r a t i o n . In c o n t r a s t , complexes where R = i - P r and t-Bu are d i s t o r t e d toward t e t r a -h e d r a l c o n f i g u r a t i o n , the in f e r e n c e i s that t h i s i s due to the presence of bulky R groups because the t-Bu complex has more t e t r a h e d r a l character than does the i s o p r o p y l complex. I t has a l s o been shown, by d i p o l e 31 29 moment measurements and o p t i c a l absorption s p e c t r a l s t u d i e s , that s a l i c y l a l d i m i n e complexes which are t r a n s - p l a n a r i n the s o l i d s t a t e appear to r e t a i n t h i s s t r u c t u r e i n s o l u t i o n . The s o l u t i o n stereochemistry of those complexes which are pseudotetrahedral as s o l i d s i s l e s s c l e a r cut and three p o s s i b i l i t i e s e x i s t . The f i r s t i s that the complexes e x i s t as an e q u i l i b r i u m mixture of planar and " t e t r a h e d r a l " isomers. The second p o s s i b i l i t y i s that the complexes d i s s o l v e without s t r u c t u r a l change and the t h i r d i s that the complexes d i s s o l v e w i t h a s t r u c t u r a l change, that i s , a change i n the d i h e d r a l angle to upon passing i n t o s o l u t i o n . The t-Bu complex f i t s i n t o the second category s i n c e i t s 29 s p e c t r a l parameters do not change upon d i s s o l u t i o n . In c o n t r a s t , complexes with R = i - P r or c y c l o h e x y l , appear to e x i s t i n s o l u t i o n as an e q u i l i b r i u m mixture of square planar and pseudotetrahedral complexes, •, j v i„ 32 28 as revealed by 1H nmr and esr s t u d i e s . The v i s i b l e s p e c t r a (d-d t r a n s i t i o n s ) i n Figure 1-5 appear as shoulders on the longer-wave l e n g t h s i d e of the intense bands i n the near-u l t r a v i o l e t except f o r Cu (t-Bu-sal)2 the d-d spectrum of which appears as a broad peak. These spectra do not lend themselves to d e t a i l e d d i s -c u s s i o n . However, one important observation concerning the three a l k y l complexes, where R = n-Bu, i - P r , and t-Bu can be made from the spectra i n Figure 1-5; namely the tendency f o r the i n t e n s i t i e s of the d-d spectra to increase i n the s p e c t r a l . o r d e r n-Bu to t-Bu wh i l e the d-d spectra s h i f t to longer wave lengths i n the same s p e c t r a l order. This tendency i s c o n s i s t e n t w i t h the dependence of the s p e c t r a l change on a d i s t o r t i o n 28 29 of the c o o r d i n a t i o n geometry towards a tetrahedron ' ; the greater the t e t r a h e d r a l d i s t o r t i o n , the greater t h i s e f f e c t . I t i s s u r p r i s i n g , t h e r e f o r e , that the spec t r a f o r the two sugar complexes [16] and [19] are not more s i m i l a r to the spectrum of the t-Bu complex, since the sugar s u b s t i t u e n t s would appear to be more bulky than the t-Bu group. Perhaps, due to the very d i f f e r e n t nature of sugar s u b s t i t u e n t s , i t i s not p o s s i b l e to compare these complexes to the simpler complexes i n which R i s an a l k y l group. However, i f the same trend of i n t e n s i t y and wave length s h i f t changes holds true w i t h i n a s e r i e s of sugar complexes, then i t appears that the complex Cu (Sug I I - s a l ) 2 [19] possesses a more square planar geometry than Cu (Sug I - s a l ) 2 [16]. Within the s e r i e s of a l k y l Cu ( R - s a l ) 2 complexes, the square planar complexes are always green or brown i n c o l o r , and the pseudotetrahedral complexes are v i o l e t . This v i o l e t c o l o r i s presumably due to the longer wave le n g t h s h i f t of the v i s i b l e absorption maxima. Therefore, the red-d i s h brown c o l o r of the Cu (Sug I - s a l ) 2 complex [16] as compared to the o l i v e green c o l o r of the Cu (Sug I I - s a l ) 2 complex [19] may support the p o s s i b i l i t y that [19] has a more square planar geometry than does [16]. Caution must be ex e r c i s e d i n t h i s i n t e r p r e t a t i o n s i n c e m e t a l — l i g a n d c h a r g e — t r a n s f e r bands may c o n t r i b u t e l a r g e l y to t h e i r c o l o r and there-f o r e comparison of the c o l o r s of sugar complexes with those of a l k y l type complexes may be i n v a l i d . The s o l i d - m u l l v i s i b l e spectrum of Cu (Sug I - s a l ) 2 [16] c o n s i s t s of three bands at 9,500, 13,500, and 20,000 cm which i s c h a r a c t e r i s t i c 20 28 29 of a pseudotetrahedral Cu (R-sal)2 complex ' ' . Therefore, the v i s i b l e a b s o r p t i o n s p e c t r a i n the s o l i d and s o l u t i o n suggests t h a t , when d i s s o l v e d , t h i s complex e i t h e r becomes square planar or loses a s i g n i f -i c a n t amount of i t s t e t r a h e d r a l character. The v i s i b l e spectrum of the cobalt sugar complex Co (Sug I - s a l ) 2 i s s t r a i g h t f o r w a r d and suggests that i t has the t e t r a h e d r a l geometry 20 expected f o r a c o b a l t ( I I ) s a l i c y l a l d i m i n e complex . Three bands are expected f o r cobalt complexes of t h i s type where v\ i s g e n e r a l l y too f a r i n the i n f r a r e d r e g i o n to be e a s i l y observed, and V2 and V3 are o f t e n found to be s p l i t i n t o s e v e r a l separate components; as a r e s u l t Co ( R - s a l ) 2 complexes g e n e r a l l y show two bands at ^ 7700 and ^ 11,200 cm assigned to components of v 2 , and a w e l l defined shoulder at ^ 17,000 -1 20 cm • The absorption bands obtained f o r the Co (Sug I - s a l ) 2 complex [18] were found at 7,300, 11,250, and 17,240 cm"1. B i s - s a l i c y l a l d i m i n e c o b a l t complexes have been found to be t e t r a h e d r a l i r r e s p e c t i v e of the nature of R and the sugar complex [18] t h e r e f o r e , seems to be no excep-t i o n . 31 ( i i ) E l e c t r o n Spin Resonance Spectroscopy Copper I I d 9 has a i e l e c t r o n s p i n which can be r e a d i l y detected by esr spectroscopy. A great deal of t h e o r e t i c a l and e m p i r i c a l i n f o r m a t i o n i s known about the esr of copper and much s t r u c t u r a l data concerning i t s complexes can be obtained from t h i s technique. The reader u n f a m i l i a r w i t h t h i s technique i s r e f e r r e d to s e c t i o n IE f o r a b a s i c d i s c u s s i o n of the esr of copper ( I I ) . B r i e f l y , though, i t i s expected that f o r copper ( I I ) i n a square planar or d i s t o r t e d t e t r a h e d r a l geometry, the room temperature s o l u t i o n spectrum w i l l c o n s i s t of four l i n e s and the 77K f r o z e n s o l u t i o n spectrum to e x h i b i t a t o t a l of eight l i n e s w i t h the four high f i e l d l i n e s c l o s e l y spaced and overlapping. This s p e c t r a l behavior was observed f o r most of the copper-sugar complexes synthesized here. The t a b u l a t i o n f o r a l l of the s p e c t r a l parameters acquired f o r the copper sugar complexes and the Cu (R-sal)2 complexes, where R= n - b u t y l , i s o p r o p y l and t - b u t y l , are shown i n Table 1-1. Here gg and ag are para-meters from the room temperature s o l u t i o n spectrum and the g„ and A„ values are from e i t h e r the frozen 77K s o l u t i o n or the s p i n - d i l u t e powder spectrum. A c l a s s i f i c a t i o n of the Cu (R-sal)2 complexes can be made on the b a s i s of esr r e s u l t s i n the same way as p r e v i o u s l y described f o r the 2 8 v i s i b l e a bsorption s p e c t r a . I t has been shown that the esr s p e c t r a l parameters of copper complexes are s e n s i t i v e to the geometry of coordin-a t i o n about the metal center. As the geometry becomes more t e t r a h e d r a l i n nature, the A„ value becomes s m a l l e r , and at the same time g,, and go begin to increase w h i l e ag decreases, as does A„. Most of the l i t e r a -t u re however, uses the changes i n A„ as a measure of t e t r a h e d r a l d i s t o r -t i o n . The Cu ( R - s a l ) 2 complexes w i t h R = n - b u t y l , i s o p r o p y l and t - b u t y l 32 TABLE I - l . Esr parameters f o r copper ( I I ) complexes Complex 80 a0 x 10~ 4 cm"1 gn A„ x 10 4 -1 cm * (a) Cu (Sug I- s a l ) 2 2.129 64.6 2.246 167. 8 * (b) Cu (Sug I I - s a l ) 2 2.130 66.6 2.259 177. 2 (c) Cu ( n - b u t y l - s a l ) 2 2.112 73.9 2.223 162. 9 (d) Cu ( i s o p r o p y l - s a l ) 2 2.119 66.3 ^2.23 VL66 * (e) Cu (t-Bu-sal)2 ^2.14 <50 2.271 135. 7 (f ) Cu (Sug I-sa l ) 2 0.5% i n Zn (Sug I- s a l ) 2 2.258 129. 7 (g) Cu (Sug I I I - s a l ) + 2.136 ^65 2.263 176. .4 (h) Cu (Sug I - s a l i c y l i c ) ( impurity) 2.30 2.278 184. ,0 * CHC&3 s o l u t i o n • powder t H 20 s o l u t i o n have esr parameters which c l e a r l y show the e f f e c t of t e t r a h e d r a l d i s t o r -t i o n on these s p e c t r a l parameters. As R changes from n-Bu to t-Bu the A values decrease and the g values increase [Table I - l ( c ) , (d) , ( e ) ] . The g,, and A,, parameters f o r the i s o p r o p y l complex are only approximate because t h i s complex e x i s t s as a mixture of geometrical isomers i n s o l u -t i o n which broadens the g,, components, making them d i f f i c u l t to measure (more w i l l be s a i d about t h i s l a t e r ) . The spectra f o r the complexes where R = a l k y l groups, were re-measured here as were the v i s i b l e absorp-t i o n s p e c t r a , so that a more accurate comparison could be made between these complexes and the sugar complexes. As was the case w i t h the v i s i b l e a b s o r p t i o n data, i t i s not c l e a r j u s t where the sugar complexes Cu (Sug I- s a l ) 2 [16] and Cu (Sug I I - s a l ) 2 [19] f a l l w i t h i n the s e r i e s of a l k y l complexes. The A values suggest that the complexes are square planar but g values put them about midway between the n - a l k y l planar and t - b u t y l pseudotetrahedral complexes. When the Zn (Sug I - s a l ) 2 complex [17] was doped w i t h 0.5% of the Cu (Sug I - s a l ) 2 [16], the powder spectrum ( f ) i n d i c a t e d a h i g h l y t e t r a h e d r a l geometry f o r the copper center. This can be explained by the f a c t that the z i n c i s almost c e r t a i n l y t e t r a -h e d r a l and when i t i s c o - c r y s t a l l i z e d w i t h a small percentage of the i s o -morphous copper complex, the l a t t e r i s forced to adopt a more t e t r a h e d r a l geometry. Both the A„ and g„ parameters are i n agreement w i t h t h i s increased t e t r a h e d r a l s t r u c t u r e and are q u i t e d i f f e r e n t from the A„ and g„ parameters obtained f o r the pure Cu (Sug I - s a l ) 2 complex. This experiment i m p l i e s that the Cu (Sug I - s a l ) 2 complex has only a small t e t r a h e d r a l d i s t o r t i o n , i f any, from a square planar geometry. The room temperature s o l u t i o n spectra and the p o l y c r y s t a l l i n e s p e c t r a f o r a l l of the complexes i n Table 1-5 are shown i n Figures 1-6 and 1-7 r e s p e c t i v e l y . As w e l l as the data presented thus f a r , a d d i t i o n a l i n f o r m a t i o n could be obtained about these complexes from d i r e c t i n s p e c t i o n of t h e i r esr s p e c t r a . The r a t e of tumbling of the complex i n s o l u t i o n ( d e s i g -nated by the c o r r e l a t i o n time x ), can a f f e c t the esr spectra dramatic-a l l y as shown i n Figure 1-6 ( a ) - ( d ) . As the molecule tumbles more and more s l o w l y , the low f i e l d l i n e s broaden to a greater extent than do the high f i e l d l i n e s ; t h i s i s because incomplete averaging of the g and A a n i s o t r o p i e s a f f e c t the low f i e l d l i n e s before the high f i e l d l i n e . This i n f o r m a t i o n can then be used to compare the s i z e of copper complexes. From the s p e c t r a i n Figure 1-6, i t i s c l e a r that the sugar complexes are much " l a r g e r " than t h e i r a l k y l counterparts. The room temperature 34 Figure 1-6. Ambient temperature chloroform s o l u t i o n esr s p e c t r a of (a) compound [16], (b) compound [19], (c) the n-butyl complex, _^ (d) the i s o - p r o p y l complex, and (e) the t - b u t y l complex at < 10 Molar.. 35 a b c d G Figure 1-7. 77K l i q u i d N2 f r o z e n s o l u t i o n esr spe c t r a of (a) compound [16], (b) compound [19], (c) the n- b u t y l complex, (d) the i s o - p r o p y l complex, (e) the t - b u t y l complex, and i n ( f ) the room temperature powder spectrum of 0.5% [16] doped i n the z i n c complex [17]. spectrum (e) of the t - b u t y l complex i s broad and poorly r e s o l v e d because the high t e t r a h e d r a l character of t h i s complex r e s u l t s i n a s h o r t e r e l e c t r o n r e l a x a t i o n time than f o r a square planar complex, which i n t u r n 31 r e s u l t s i n broadened t r a n s i t i o n s The p o l y c r y s t a l l i n e s p e c t r a i n Figure 1-7 a l s o c o n t a i n a d d i t i o n a l i n f o r m a t i o n ; i n s o f a r that they a l l have an a x i a l type of l i n e shape t h i s i m p l i e s that the copper center possesses an a x i a l , or near a x i a l , sym-metry. Spectrum (d) of the i s o p r o p y l complex a l s o r e v e a l s that a mix-ture of geometries are present f o r t h i s molecule i n s o l u t i o n . This deduction i s based on the observation of the broad g„ components i n t h i s 28 spectrum which have been p r e v i o u s l y a t t r i b u t e d to overlapping l i n e s of a mixture of planar and pseudotetrahedral species. Computer simu l a -28 t i o n of the spectrum showed that t h i s was a reasonable assignment. This has an important c o r o l l a r y i n that i t now seems safe to assume from the s p e c t r a i n Figure 1-7 that the other sugar- and alkyl-complexes e x i s t as a s i n g l e s t r u c t u r a l species i n s o l u t i o n . Both the room temperature s o l u t i o n spectrum and the 77K spectrum of the water s o l u b l e complex Cu (Sug I l l - s a l ) [21] are shown together i n Figure 1-8. The room temperature spectrum (a) r e v e a l s that the molecule must be tumbling r e l a t i v e l y s l o w l y s i n c e the low f i e l d l i n e s are very broad. This suggests that the complex i s l a r g e and t h e r e f o r e i m p l i e s that the complex does c o n t a i n the sugar moiety. In t h i s spectrum, how-ever, i t appears as though there are two overlapping sets of resonances s i n c e the ao h y p e r f i n e c o u p l i n g values are not a l l the same. This sug-gests that the compound i s e i t h e r a mixture of complexes or a mixture of geometric isomers of the same complex. The 77K spectrum i s an a x i a l type of spectrum but the r a t h e r broad Figure 1-8. (A) the aqueous room temperature esr spectrum and (B) the 77K frozen aqueous esr spectrum of compound [21]. LO 38 f e a t u r e l e s s g„ components again suggest a mixture of complexes or complex • geometries, as was imp l i e d by the room temperature spectrum. Thus although the exact s t r u c t u r e of t h i s complex i s not unequivocally estab-l i s h e d by these sp e c t r a alone, they are c e r t a i n l y c o n s i s t e n t w i t h the s t r u c t u r e proposed i n I B ( i i ) . The u n d i l u t e d powder spectrum of the proposed b i n u c l e a r copper com-plex Cu2(Sug I - s a l i c y l i c ) 2 [23] i s shown i n Figure 1-9. I t i s b e l i e v e d that t h i s spectrum i s not of the b i n u c l e a r complex i t s e l f , but i s i n s t e a d of a tr a c e of a mononuclear copper complex i m p u r i t y , d i l u t e d w i t h the b i n u c l e a r complex. This i n t e r p r e t a t i o n seems reasonable s i n c e (a) the powder spectrum of a pure mononuclear copper complex i s expected to be h i g h l y exchange broadened, which i s not observed, and (b) the r e c e i v e r gain of the instrument had to be set to a very high l e v e l i n order to see the spectrum, which i m p l i e s that the molecule g i v i n g r i s e to the spectrum i s at a very low c o n c e n t r a t i o n . That t h i s spectrum c l e a r l y shows n i t r o g e n super-hyperfine s t r u c t u r e proves that t h i s mononuclear complex contains the sugar l i g a n d . The reason why the esr spectrum of the b i n u c l e a r complex i t s e l f i s not detected i s because exchange c o u p l i n g between the two copper ions produce s i n g l e t (S = 0) and t r i p l e t (S = 1) e l e c t r o n i c s t a t e s . Since the s i n g l e t s t a t e i s expected to have zero magnetic moment only molecules i n the t r i p l e t s t a t e can give r i s e to an e s r s i g n a l . Two scenarios are then p o s s i b l e to e x p l a i n the l a c k of a dete c t a b l e esr spectrum. The f i r s t i s that J > kT where J i s the energy d i f f e r e n c e between the s i n g l e t and t r i p l e t s t a t e s , k i s the Boltzman constant and T i s the temperature. This would ensure that only the S = 0 s t a t e was populated and the molecule would be diamagnetic. The second p o s s i b i l i t y i s that J ^ kT but that the energy s p l i t t i n g 39 Figure 1-9. The 77K frozen powder esr spectrum of the mononuclear copper complex im p u r i t y w i t h i n the b i n u c l e a r complex [23]. between the three mg s t a t e s + 1 , 0, -1, o r i g i n a t i n g from the t r i p l e t S = 1 s t a t e , i s too l a r g e f o r s p i n t r a n s i t i o n s to be induced by micro-wave energy. The way to d i s t i n g u i s h between these two p o s s i b i l i t i e s would be to determine the magnetic moment of the pure compound; t h i s has not been done at t h i s time. The existence of the b i n u c l e a r copper complex i s a l s o s t r o n g l y supported by the mass spectrum as w i l l be d i s -cussed l a t e r i n I - C ( i v ) . ( i i i ) Magnetic Moments Often a s s o c i a t e d w i t h red s p e c t r a l s h i f t s , as seen i n ( i ) f o r the a l k y l complexes of Cu (R-Sal)2 w i t h pseudotetrahedral s t r u c t u r e s , are l a r g e r magnetic moments; a y e£f value of _> 1.89 BM has been considered 31 c h a r a c t e r i s t i c of non-planar geometry . This does not, however, appear to be a r e l i a b l e s t r u c t u r a l c r i t e r i o n ; f o r example, recent measure-31 ments of s o l i d Cu ( i - P r - s a l ) 2 and Cu (t-Bu-sal)2 which are known to be pseudotetrahedral, have y i e l d e d values of 1.84 and 1.83 BM r e s p e c t i v e l y 31 compared to 1.90-1.93 BM obtained e a r l i e r . The magnetic moments c a l -c u l a t e d from the esr and Faraday methods, f o r the copper complexes shown p r e v i o u s l y , are given i n Table 1-2. I n i t i a l l y i t was thought that the change i n V^^, f o r the s o l i d complex Cu (Sug I - s a l ) 2 (1.91 BM)*, upon d i s s o l u t i o n of the s o l i d r e f l e c t e d a change i n geometry. However, i n l i g h t of the recent data c i t e d above f o r Cu ( i - P r - s a l ) 2 and Cu (t-Bu-sal) i t does not seem wise to be too dogmatic on t h i s c o n c l u s i o n . The s o l u t i o n magnetic moments were c a l c u l a t e d from gQ by the r e l a t i o n s h i p y e f f = g 0 /S(S + 1) *0bt sined by Dir. R. C • Thompson of t h i s departnGnt • TABLE 1-2. Magnetic moments of copper complexes 41 u f , from esr u f f Faraday Complex BM 6 BM (a) Cu (Sug I - s a l ) 2 [16] 1.84 1.91 (b) Cu (Sug I I - s a l ) 2 [19] 1.84 (c) Cu ( n - B u - s a l ) 2 1.83 (d) Cu ( i - P r - s a l ) 2 1.84 (e) Cu ( t - B u - s a l ) 2 1.85 (f ) Cu (Sug I I I - s a l ) 2 [21] 1.85 (g) Mononuclear i m p u r i t y w i t h i n b i n u c l e a r complex [23] 1.99 where S i s i f o r copper ( I I ) . These data are a l l very s i m i l a r (1.83-1.85 BM) although there i s a s l i g h t trend i n going from R = n-Bu to t-Bu. Since i t was not p o s s i b l e to o b t a i n go f o r the mononuclear com-pl e x impurity w i t h i n the b i n u c l e a r complex [23] a value was estimated by s u b s t i t u t i n g a value of g±, (2.03) derived from the spectrum i n Figure 1-9, i n the f o l l o w i n g equation (see s e c t i o n IE) g 0 = 1/3 (g„ + 2 g_,) = 2.30 ( i v ) Mass Spectrometry Copper has two isotopes ( S 3Cu, 69.09% and 5 5 C u , 30.91%) which con-f e r defined i s o t o p e - p a t t e r n s to the mass s p e c t r a l fragmentation of copper-containing complexes and thereby make t h i s a u s e f u l t o o l f o r s t r u c t u r e determination. A simple i l l u s t r a t i o n of how the isotope p a t t e r n can be c a l c u l a t e d f o r a b i n u c l e a r copper complex i s as f o l l o w s . F i r s t l y , f o r a s i n g l e copper atom the M and M + 2 peaks w i l l be i n a r a t i o ca 7:3. With the M 7 M * 2 3 63r Cu Cu C O c o second copper atom there are three p o s s i b l e combinations: D 3 C u D 3 C u : 6 3 C u 6 5 C u : 6 5 C u 6 5 C u which w i l l have r e l a t i v e i n t e n s i t i e s i n the r a t i o of 49:42:9 r e s p e c t i v e l y because of the same 7:3 r a t i o . When the carbon M 7 M * 2 3 63 Cu 4 9 65 Cu ! " x N M * 4 4 2 i i 9 I 63Cu63Cu " c ^ C u 6 5Cu 6 5Cu isotope e f f e c t ( 1 3C, 1.08%) i s taken i n t o account f o r a molecule which has 42 carbon atoms, the isotope p a t t e r n i s as shown i n Figure 1-10. This s i m u l a t i o n gives e x a c t l y the same molecular i o n p a t t e r n as that obtained e x p e r i m e n t a l l y , and t h i s evidence along w i t h the parent mass of 1056, provides concrete evidence f o r the ex i s t e n c e of the b i n u c l e a r copper complex [23]. Obviously, such isotope patterns are a l s o very u s e f u l f o r c h a r a c t e r i z i n g mononuclear copper complexes, f o r which the peak r a t i o s are approximately 100:20:40:10 f o r the M, M + 1, M + 2, and M + 3 peaks r e s p e c t i v e l y , depending upon the number of carbon atoms 43 100 46 10 63 63 Cu Cu B 86 3 9 9 _1_ 63 65 Cu Cu 100 9 6 18 8 _L o 65 65 _L_ Cu Cu D U6 39 2 7 M M *1 M * 2 M * 3 M * 4 M * 5 M * 6 Figure 1-10. Si m u l a t i o n of the mass s p e c t r a l i sotope p a t t e r n f o r a b i n u c l e a r copper complex. 44 i n v o l v e d . The mass spectrum of a l l the mononuclear copper sugar com-plexes studied here show t h i s p a t t e r n . In the mass spectrum of the water s o l u b l e sugar complex [21] there i s a parent peak corresponding to the molecular weight w i t h the c o r r e c t isotope p a t t e r n , but some addi-t i o n a l peaks are found as high as 420 amu. Since t h i s complex decomposes upon heating to 150°C t h i s may be re s p o n s i b l e f o r the observed high mass peaks. C l e a r l y t h i s complex i s not of the b i s - t y p e f o r which a parent peak at 627 amu would be expected. Zinc has four n a t u r a l abundance isotopes ( 6 1 +Zn, 48.89%; 6 5 Z n , 27.81%; 6 7 Z n , 4.11%; and 6 8 Z n , 18.57%) which confer w e l l defined i s o t o p e - p a t t e r n s to the mass spectrum of z i n c complexes. For a mole-cule w i t h 40 carbon atoms, using the same method of c a l c u l a t i o n a p p l i e d to the copper case, the r a t i o s f o r the M:M+l:M+2:M+3:M+4:M+5:M+6 peaks are approximately 100:44:65:33:46:18:3. This r a t i o p a t t e r n i s indeed seen i n the mass spectrum of the zinc-sugar complex [17], thus confirming i t s s t r u c t u r e . (v) Nmr Spectroscopy Proton nmr spectroscopy can, very o f t e n , be a p p l i e d to paramag-n e t i c complexes, but the spec t r a are u s u a l l y much more d i f f i c u l t to i n t e r p r e t than f o r diamagnetic complexes. The r e s o l u t i o n of such s p e c t r a are l a r g e l y dependent upon the metal in v o l v e d and the coordina-33 t i o n geometry about the metal . The l i n e widths are dependent f o r the most part upon the r e l a x a t i o n time, T-^ e> of the unpaired e l e c t r o n which i n t u r n i s dependent upon the c o o r d i n a t i o n geometry about the copper atom. The shor t e r T, the narrower the l i n e w i d t h s become and i t i s l e known that copper complexes w i t h a square planar geometry have a longer T, than complexes w i t h a more t e t r a h e d r a l c o o r d i n a t i o n . Therefore l e s p e c t r a of Cu (R-sal)2 complexes where R = t-Bu and the geometry i s pseudotetrahedral, have narrower l i n e widths than complexes where R 32 i s an unbranched a l k y l group and the geometry square planar . This e f f e c t has been used to study the p o s s i b i l i t y of a s t r u c t u r a l e q u i l i -brium between square planar and pseudotetrahedral as proposed f o r the 32 complex Cu ( i - P r - s a l ) 2 • For t h i s complex i t has been found that as the temperature i s r a i s e d the proton l i n e widths become narrower. This has been a t t r i b u t e d to an increased population of the pseudotetrahedral species at high temperature which r e s u l t s i n a s h o r t e r T^g and t h e r e f o r e , a narrower l i n e width. Copper ( I I ) complexes i n a square planar or pseudotetrahedral geometry are e s s e n t i a l l y a b o r d e r l i n e case and although the proton resonances are very broad, some of them can be detected. In the case of the sugar complex Cu (Sug I - s a l ) 2 (Figure 1-11) only the acetate protons of the sugar r i n g s can be observed c l e a r l y , and the other very broad peaks cannot be e a s i l y assigned. Cobalt ( I I ) t e t r a h e d r a l complexes have the r e q u i r e d short T^g f o r the observation of w e l l r e s o l v e d nmr spectra but u n f o r t u n a t e l y , due to time l i m i t a t i o n s , the c o b a l t sugar complex [18] was not examined by nmr. 33 N i c k e l ( I I ) complexes of t h i s type have been stud i e d e x t e n s i v e l y by JH nmr p r i n c i p a l l y because the s p e c t r a of these complexes have narrow l i n e widths and are very w e l l r e s o l v e d . This can be a t t r i b u t e d to the f a c t that these n i c k e l complexes e x i s t as an e q u i l i b r i u m mixture of diamagnetic (square planar) and paramagnetic ( t e t r a h e d r a l ) s t r u c t u r e s . However, even though sharp resonances can be observed i n the s p e c t r a of s a l i c y l a l d i m i n e n i c k e l ( I I ) complexes, assignment i s not t r i v i a l s ince the usual chemical s h i f t o r d e r i n g commonly known f o r diamagnetic compounds OAc Figure 1-11. Proton nmr spectrum (270 MHz) of the copper sugar complex [16] i n deuteriochloroform. 47 does not h o l d f o r these paramagnetic complexes. The z i n c ( I I ) complexes, s i n c e they are diamagnetic, can be r e a d i l y observed by nmr spectroscopy and the 1H nmr sp e c t r a of the z i n c sugar complex Zn (Sug I - s a l ) 2 [17], along w i t h the parent l i g a n d [8] are shown i n Figure 1-12. The spectrum of the z i n c complex shows some f r e e l i g a n d i m p u r i t y which could not be removed by r e c r y s t a l l i z a t i o n . I t i s d i f f i -c u l t to say whether t h i s a r i s e s from decomposition of the complex i n s o l u t i o n , or whether i t i s i n t r i n s i c a l l y present i n the sample. Since the C:H:N r a t i o s are s i m i l a r f o r both the l i g a n d and complex, elemental a n a l y s i s w i l l not r e v e a l a small percentage (< 10%) of the l i g a n d i m p u r i t y . The presence of the z i n c atom disperses the aromatic r e g i o n very n i c e l y i n t o two t r i p l e t s and two doublets and a l s o s h i f t s most of the sugar r i n g protons to high f i e l d compared w i t h the parent l i g a n d . Apart from the observation of these s h i f t s , nothing more can be con-cluded from the spectrum of the z i n c complex. ID: Other Chemistry For s a l i c y l a l d i m i n e complexes i n general, by f a r the most abundant and thoroughly s t u d i e d are the n i c k e l complexes. A l l attempts to form n i c k e l complexes from the sugar l i g a n d s and n i c k e l a c e t a t e , however, f a i l e d . Some of these attempted r e a c t i o n s are worth mentioning here, though. Reaction of the sugar s a l i c y l a l d i m i n e s [ 6 ] , [ 8 ] , [10], and [11] i n the u sual way w i t h n i c k e l ( I I ) acetate i n e i t h e r methanol or ethanol f a i l e d to give any product and i n a l l cases the s t a r t i n g sugar l i g a n d could be recovered. . I f e i t h e r the amino sugars [7] or [9] were reacted w i t h the preformed s a l i c y l a l d e h y d e complex, as shown below, the J CDCl: AROMATIC / j u H 3 H 4 HT H 6 ' JIJ IjJLnLJ OMe H, OAc 2 T M S J l 1 0 i • • • • i • • • • ' Figure 1-12. Proton nmr spectrum (270 MHz) of the z i n c complex [17] (A) and the l i g a n d [8] (B). 49 i-OAc 0 R + NH2 [7] R=0Me [9] R=0Ac [8] R=OMe [10] R=OAc s a l i c y l a l d i m i n e S c h i f f ' s bases [8] and [10] were i s o l a t e d from the r e a c t i o n mixture. Obviously the amino group i s condensing w i t h the carbonyl of the s a l i c y l a l d e h y d e complex, as i t should, but the n i c k e l i o n i s being e x p e l l e d , suggesting that these complexes are simply not s t a b l e . The analogous c y c l o h e x y l n i c k e l complex, however, can be synthesized r e a d i l y e i t h e r by r e a c t i o n of the s a l i c y l a l d i m i n e w i t h n i c k e l acetate or by r e a c t i o n of cyclohexylamine w i t h the preformed 20 s a l i c y l a l d e h y d e - n i c k e l complex I t was then reasoned that the sugar s u b s t i t u e n t s might have a r e p e l l i n g e f f e c t on the n i c k e l i o n and th e r e f o r e the complexing u n i t was moved f u r t h e r away from the sugar r i n g by means of a benzene s u l -phonamide spacer group, as shown i n the f o l l o w i n g r e a c t i o n scheme. However, r e a c t i o n of the s a l i c y l a l d i m i n e [28] w i t h n i c k e l ( I I ) acetate r e s u l t e d i n cleavage of the S c h i f f ' s base C = N double bond and forma-t i o n of the s a l i c y l a l d e h y d e n i c k e l complex along w i t h the f r e e amine 50 compound [27]. This cleavage could not be prevented even i f sodium acetate was used to make the r e a c t i o n s l i g h t l y b a s i c . Reaction of t h i s l i g a n d [28] w i t h c u p r i c acetate was not attempted. IE: E l e c t r o n Spin Resonance Spectroscopy of Copper ( I I ) ( i ) General Esr E l e c t r o n s p i n resonance spectroscopy occurs i n a paramagnetic mole-cu l e when t r a n s i t i o n s between the Zeeman l e v e l s , whose degeneracy may be l i f t e d by the a p p l i c a t i o n of a magnetic f i e l d H, are induced by an 51 electromagnetic f i e l d Hi of frequency v. The s e p a r a t i o n of these Zeeman l e v e l s i s described i n (1) E = hu = gBH (1) where h i s Plank's constant, B the Bohr magneton (en/2m), m the mass of the e l e c t r o n , and g the Lande s p l i t t i n g f a c t o r , a dimentionless parameter r e l a t e d to the e f f e c t i v e magnetic moment of the e l e c t r o n by y e = - gBS (2) where Sh i s the s p i n angular momentum v e c t o r . D i f f e r e n c e s i n the Zeeman energy between d i f f e r e n t molecules r e s u l t i n changes i n g from i t s f r e e e l e c t r o n s p i n - o n l y value of 2.00232, as a r e s u l t of s p i n - o r b i t c o u p l i n g . Thus the g value i s used to c h a r a c t e r i z e the p o s i t i o n of the resonance i n the frequency spectrum. As shown i n equation (1) the resonance frequency i s dependent upon the a p p l i e d f i e l d ; most experiments, i n c l u d i n g the ones described h e r e i n , are conducted at X-band (about 9.5 GHz), which corresponds to an e x t e r n a l f i e l d of the order of 2.5-3.5 KG. In the experiments described here, net a b sorption of microwave energy from Hi occurs at resonance as a r e s u l t of the greater p r o p o r t i o n of spins present i n the lower energy s t a t e . ( i i ) Copper ( I I ) Esr This s e c t i o n has been adapted i n part from the M.Sc. t h e s i s w r i t t e n 34 by C a r l A H e y n e and from reviews on t r a n s i t i o n metal esr For the d 9 c o n f i g u r a t i o n of copper ( I I ) the e f f e c t i v e e l e c t r o n S = \ and the s p i n angular momentum mg = ± \ gives r i s e to a doubly degenerate s p i n energy s t a t e , the degeneracy of which i s removed when an e x t e r n a l magnetic f i e l d i s a p p l i e d . The lower energy s t a t e has the s p i n a l i g n e d w i t h the e x t e r n a l f i e l d corresponding to m = - w h i l e the high energy s t a t e , m = + i , has i t s s p i n opposed to the f i e l d . A t r a n s i t i o n between the two s t a t e s occurs upon absorption of microwave energy as given by (1) E = hv = ggH (1) As w i l l be seen f o r n i t r o x i d e s l a t e r i n s e c t i o n H I E , the qu a n t i t y g i s dependent upon the e f f e c t i v e o r i e n t a t i o n of the molecule c o n t a i n i n g the unpaired e l e c t r o n w i t h respect to the magnetic f i e l d . I f the copper i o n i s l o c a t e d i n a p e r f e c t l y cubic c r y s t a l s i t e , the g value i s indepen-dent of the o r i e n t a t i o n of the c r y s t a l and i s s a i d to be i s o t r o p i c . In a c r y s t a l s i t e of lower symmetry, both the g value and the s p l i t t i n g value A are o r i e n t a t i o n dependent and are s a i d to be a n i s o t r o p i c . The z - d i r e c t i o n i s defined as co i n c i d e n t w i t h the h i g h e s t - f o l d r o t a t i o n a x i s . In a x i a l l y symmetric systems the g and A values are give the n o t a t i o n g x x = 8yy = 8-L ( 3 ) g = 8, zz (4) AXX " A y y = A l ( 5 ) A z z = A " ( 6 ) When the unpaired e l e c t r o n of the Cu ( I I ) i o n couples with the nuclear s p i n of I = 3/2 the absorption i s s p l i t i n t o 2 1 + 1 components. The cause of t h i s " h y p e r f i n e " coupling i n t e r a c t i o n a r i s i n g mainly from the Fermi contact term. Thus f o r 1 = 3 / 2 the esr spectrum (Figure 1-13) c o n s i s t s of four l i n e s . The s e l e c t i o n r u l e s are Am^ = ± 1 and Am^ . = 0. For the s p e c i a l case where there are two copper ions i n the same molecule which are exchange coupled, the energy l e v e l diagram i s q u i t e d i f f e r e n t (Figure 1-14). Now there i s a s i n g l e t S = 0 and a t r i p l e t S. = 1 e l e c -t r o n i c s t a t e which are separated by an energy d i f f e r e n c e J . The S = 1 s t a t e i s s p l i t i n t o three non degenerate m^  s t a t e s + 1 , 0, -1 when a Figure 1-13. Spin s t a t e energy l e v e l diagram f o r a copper ( I I ) n u c l e i i n a magnetic f i e l d . 54 Figure 1-14. Energy l e v e l diagram f o r two i n t e r a c t i n g copper ( I I ) n u c l e i i n a magnetic f i e l d . 55 magnetic f i e l d i s a p p l i e d . Although not shown, there are a l s o super-imposed on these m„ s t a t e s , seven m s t a t e s r e s u l t i n g from the hyperfine i n t e r a c t i o n of the e l e c t r o n w i t h the two S = 3/2 spins of the Cu ( I I ) n u c l e i . Therefore, only e l e c t r o n s i n the S = 1 s t a t e can give r i s e to a two l i n e esr spectrum and t h i s mole f r a c t i o n i s dependent upon the Botzman d i s t r i b u t i o n which i s dependent upon the energy d i f f e r e n c e J and the temperature. No other d e t a i l s w i l l be given here about such b i n u c l e a r s p e c t r a . Hence the room temperature spectrum of a mononuclear copper complex c o n s i s t s of four l i n e s due to the copper nuclear hyp e r f i n e i n t e r a c t i o n . The band shapes are dependent upon the tumbling r a t e or c o r r e l a t i o n time T c . I f the molecule tumbles r a p i d l y enough the a n i s o t r o p i e s i n g and A w i l l be averaged, whereas i f the r a t e i s slow, l i n e broadening w i l l occur. Since the g„ low f i e l d m^. = + 3/2 l i n e must average with a d i s t a n t g_, high f i e l d m = - 3/2 l i n e (Figure 1-15), t h i s component t h e r e f o r e broadens f a s t e s t as the r a t e of tumbling decreases. In a p o l y c r y s t a l l i n e sample, or 77K frozen s o l u t i o n , the g and A a n i s o t r o p i e s are not averaged at a l l . The molecular axes of symmetry are randomly d i s t r i b u t e d and the observed resonance l i n e shape repre-sents the s u p e r p o s i t i o n of the i n d i v i d u a l resonances. The d e r i v a t i v e spectrum shows a weak set of l i n e s at g„, corresponding to those molecules w i t h t h e i r symmetry axes p a r a l l e l to the a p p l i e d f i e l d and a set of strong l i n e s at g^ corresponding to those molecules w i t h the symmetry axes perpendicular to the a p p l i e d f i e l d . This spectrum i s depicted i n the s t i c k diagram of Figure 1-15. This type of spectrum was seen i n Figure 1-7 where only three of the four g„ l i n e s are seen and the g, l i n e s are 56 Figure 1-15. Schematic r e p r e s e n t a t i o n of the esr spectrum of copper ( I I ) showing both g„ and gx components. seen only as a s i n g l e broad l i n e . Superhyperfine s t r u c t u r e can sometimes be seen, as i n Figure 1-9, which r e s u l t from the h y p e r f i n e i n t e r a c t i o n of the s p i n 1 n i t r o g e n nucleus and the e l e c t r o n s p i n . The 2 1 + 1 r u l e holds here again and t h e r e f o r e the number of superhyperfine l i n e s depends upon the number of n i t r o g e n donor atoms coordinated to the copper i o n . Because the copper hy p e r f i n e l i n e s are narrower at high f i e l d , the n i t r o g e n superhyperfine l i n e s are more c l e a r l y resolved on the high f i e l d s i d e of the spectrum, although they can sometimes a l s o be seen on the g„ components. For Table 1-1 the A and g values were abstracted d i r e c t l y from the f i e l d - c o r r e c t e d s p e c t r a . The A„ values were a l l taken as the separa-t i o n between the + i and - £ l i n e s and the ag values were determined from the - i, - 3/2 separation. Both g„ and g 0 were taken as the center po i n t between the + i and - i l i n e s . The values of hype r f i n e constants obtained by t h i s procedure are i n u n i t s of magnetic f i e l d and can be con-verted i n t o frequency u n i t s by the equation A(cm "S = A(gauss) x x 9.3484 x 10 (7) S t y » i r 5 I f d e s i r e d , g, and A, can be obtained from the equations g 0 = l/3(g„ = 2g_,); a 0 = 1/3(A„ + 2A_,) (8) The esr spe c t r a can a l s o be used to c a l c u l a t e s o l u t i o n magnetic moments f o r these complexes. The f o l l o w i n g equations r e l a t e y f f a n d S O V e f f = y s 0 ( 1 " 2 K 2 Xo/10Dq) (9) where y n i s the s p i n only magnetic moment, K i s the o r b i t a l r e d u c t i o n s 0 f a c t o r , and X 0 i s the s p i n o r b i t c o upling constant which f o r the f r e e io n equals - 829 cm \ g = g e ( l - 2K 2 X0/10Dq) (10) y e f f = g 0 /S(S + 1) (11) Using equation (11) and s u b s t i t u t i n g S = i f o r Cu ( I I ) and the go values obtained from the room temperature s o l u t i o n s p e c t r a , we can compare y ef£ c a l c u l a t e d from esr data and y g££ obtained from bulk magnetic s u s c e p t i b i l i t y measurements as shown e a r l i e r i n s e c t i o n I C ( i i i ) IF: Summary and Conclusions Compounds [ 6 ] , [ 8 ] , and [10] have been known f o r over f i f t y years and [11] i s a new S c h i f f ' s base sugar compound; a l l were prepared r e a d i l y i n high y i e l d . I t has been shown by nmr spectroscopy that f o r compounds [ 8 ] , [10], and [11] hydrogen bonding e x i s t s between the aromatic hydroxyl proton and the n i t r o g e n atom from the sugar group. Hydrogen bonding being a p r e r e q u i s i t e f o r m e t a l - c h e l a t i o n by these l i g a n d s , i t was not unexpected that these compounds formed sugar metal complexes; however, t h i s only occurred w i t h c e r t a i n metal i o n s . Ligand [8] formed copper, cobalt and z i n c ( I I ) complexes, whereas l i g a n d s [ 6 ] , [10], and [11] only complexed w i t h copper ( I I ) . None of these l i g a n d s formed complexes w i t h n i c k e l ( I I ) . The z i n c and c o b a l t complexes of [8] were a l s o found to be q u i t e unstable i n s o l u t i o n and even the copper complexes of [8] and [10] d i s s o c i a t e d to the parent l i g a n d when run on s i l i c a g e l t i c . I t i s not understood why n i c k e l complexes of the sugar l i g a n d s could not be prepared. Since the t e t r a h e d r a l z i n c and c o b a l t complexes [17] and [18] were l e s s s t a b l e than the copper analogue [16], i t could be reasoned that metals which p r e f e r a t e t r a h e d r a l geometry might not form complexes w i t h these l i g a n d s . N i c k e l ( I I ) , however, can r e a d i l y adopt e i t h e r a square planar or t e t r a h e d r a l c o o r d i n a t i o n and t h e r e f o r e t h i s argument does not provide an answer. A v a r i e t y of spectroscopic and p h y s i c a l techniques have been used to c h a r a c t e r i z e the sugar-metal complexes synthesized. V i s i b l e absorp-t i o n spectroscopy and e l e c t r o n s p i n resonance spectroscopy both provide s t r u c t u r a l i n f o r m a t i o n concerning c o o r d i n a t i o n geometry as w e l l as proof of e x i s t e n c e . However, n e i t h e r technique gave unequivocal i n f o r m a t i o n w i t h regards to the exact c o o r d i n a t i o n geometry of the copper ( I I ) com-plexes [16] and [19]. When compared to a s e r i e s of analogous a l k y l complexes of known s t r u c t u r e , v i s i b l e absorption, spectroscopy suggested that the copper sugar complexes [16] and [19] had e s s e n t i a l l y square planar geometry w i t h compound [16] being s l i g h t l y more d i s t o r t e d towards t e t r a h e d r a l symmetry than was [19]. The s o l i d m u l l s p e c t r a of [16], however, suggested a pseudotetrahedral s t r u c t u r e f o r [16] i n the s o l i d . I t was obvious from the l i t e r a t u r e that esr spectroscopy was by f a r the most powerful p h y s i c a l technique which could be used to determine the s t r u c t u r e of the copper complexes synthesized here. Thus i t can i n d i -cate whether there i s a copper i o n present, how f a s t i t i s tumbling i n s o l u t i o n from the r e l a t i v e l i n e shapes and hence the approximate s i z e of the molecule, the c o o r d i n a t i o n geometry from g and A parameters, and whether the l i g a n d contains a n i t r o g e n donor atom from superhyperfine i n t e r a c t i o n s . The esr data obtained i n t h i s study provide evidence, from A v a l u e s , of square planar geometry f o r [16] and [19], but g values gave c o n f l i c t i n g evidence f o r moderate t e t r a h e d r a l d i s t o r t i o n i n these complexes. The esr powder spectrum of the copper-doped z i n c complex gave the g„ and A„ values expected f o r a s i g n i f i c a n t t e t r a h e d r a l d i s t o r --4 -1 t i o n . The A,, value changed from 168 * 10 cm f o r the pure complex -4 -1 [16] to 129 x 10 cm f o r the copper-doped z i n c complex, w h i l e the g„ values changed from 2.246 to 2.258. The change i n g„ was expected to be much l a r g e r than that found and t h e r e f o r e i t i s f e l t that changes i n A values may be a b e t t e r c r i t e r i o n f o r comparing the c o o r d i n a t i o n geometry between s i m i l a r types of complexes. The z i n c - d i l u t i o n experiment a l s o p o i n t s out that the copper complex [16] could not have a large t e t r a -h e d r a l d i s t o r t i o n i n s o l u t i o n . The bulk of the data, t h e r e f o r e , suggests that the copper complexes [16] and [19] possess square planar geometries i n s o l u t i o n , or have only a s l i g h t t e t r a h e d r a l d i s t o r t i o n . Magnetic moments, mass s p e c t r a , and nmr spectra of the copper com-plexes are a l l c o n s i s t e n t w i t h the proposed s t r u c t u r e s . Mass sp e c t r o -metry was found to be p a r t i c u l a r l y u s e f u l i n determining the s t r u c t u r e of the b i n u c l e a r complex [23] s i n c e the isotope patterns f o r mono- and b i -nuclear copper complexes are e a s i l y d i s t i n g u i s h e d and very d i a g n o s t i c . The water s o l u b l e glucosamine complex [21] seems to have a d i f f e r e n t s t r u c t u r e than the normal s a l i c y l a l d i m i n e copper complexes. Both mass spectrometry and elemental a n a l y s i s suggest that t h i s complex has only one sugar s a l i c y l a l d i m i n e u n i t w i t h the t h i r d c o o r d i n a t i o n s i t e taken up by a hydroxyl group from the sugar r i n g and the f o u r t h by a water molecule. C l e a r l y , from the standpoint of p o t e n t i a l pharmaceutical a p p l i c a -t i o n s , water s o l u b l e sugar complexes such as the one j u s t described [21] are by f a r the most important. Future c o n t i n u a t i o n of the work i n t h i s chapter might best i n v o l v e attempts to synthesize more of these water s o l u b l e sugar complexes, employing a wide v a r i e t y of sugars, l i g a n d m o i e t i e s , and metal ions. 61 References 1. J . A. Rendleman, J r . , Advances i n Carbohydrate Chemistry, 21, 209(1966). 2. S. J . Angyal, Pure and Applied Chemistry, 35, 131(1973). 3. S. J . Angyal, C. L. Bodkin, J . A. M i l l s and P. M. P o j e r , Aust. J . Chem., 30, 1259(1977). 4. Proceedings of Symposium on Development of Iron Chelators f o r C l i n i c a l Use, U.S. Department of Healt h , Education, and Welfare, eds. W. F. Anderson and M. C. M i l l e r , 1975. 5. German Patent No. 564437, 1932. 6. M. M i y a z a k i , S. Nishimura, A. Yoshida and N. Okubo, Chem. Pharm. B u l l . , _27, 532(1979). 7. B. R. James, Chemistry i n Canada, December, 13(1978); S. R. Landor, B. J . M i l l e r and A. R. T a t c h e l l , J . Chem. Soc. (C), 197(1967). 8. S. Hannessian and G. P a t i l , Tetrahedron L e t t e r s , 1031(1978); J . J . Wright, A. Cooper, P. J . L. D a n i e l s , T. L. Nagabushan, D. Rome, W. N. Turner and J . Weinstein, J . A n t i b i o t i c s , 29, 714(1976). 9. I . Tabushi, N. Shimizu, T. Sugimoto, M. Shiozuka, K. Yamamura, J . Am. Chem. S o c , 99, 7100(1977). 10. Yu. A. Zhdanov, 0. A. Osipov, V. P. G r i g o r i e v , A. D. Garnovski, Yu. E. Alexeev, V. G. Alexeeva, N. M. Gontmacher, P. A. Perov, V. G. Z a l i o t o v , V. N. Fomina, T. A. Useman, 0. N. Nechaeva and V. N. Mirny, Carbohydrate Research, 38, Cl(1974). 11. W. R. C u l l e n and Y. Sugi, Tetrahedron L e t t e r s , 1635(1978). 12. F. R. Ha r t l e y and P. N. Vezey, Advances i n Organometallic Chemistry, 15, 189(1977). 13. M. Z. Elsabee, M. Matlar and G. M. Habashy, J . Poly. S c i . , 14_, 1773(1976). 14. V. Gibb and L. D. H a l l , Carbohydrate Research, 55, 239(1977). 15. V. Gibb and L. D. H a l l , i b i d . , 63, Cl(1978). 16. L. D. H a l l , P. R. S t e i n e r and D. C. M i l l e r , Can. J . Chem., 57, 38(1979). 17. A. M. Slee, M.Sc. t h e s i s , U n i v e r s i t y of B r i t i s h Columbia, 1973. 62 18. B. Lonnerdal, J . Carlsson and J . Porath, FEBS. L e t t e r s , 75, 89(1977); J . P. Lebreton, i b i d . , 80, 351(1977). 19. I. Armitage and L. D. H a l l , Carbohydrate Research, 24, 221(1972); D. Horton and J . D. Wander, i b i d . , 39, 141(1975); L. D. H a l l and C. M. Pre s t o n , i b i d . , 41, 53(1975); B. Casu, G. G a t t i , N. Cyr and A. S. P e r l i n , i b i d . , 41, C6(1975); S. J . Angyal, i b i d . , 2^ 6, 271(1973); R. F. Butterworth, A. G. Pernet and S. Hannessian, Can. J . Chem., 4j), 981(1971); P. G i r a r d , H. Kogan and S. David, B u l l . Soc. Chim. France, 12, 4515(1970). 20. R. H. Holm, G. W. E v e r e t t , J r . , and A. Chakravorty, Progr. Inorg. Chem. , ]_, 83(1966) . 21. D. Horton, The Amino Sugars, ed. R. W. Jeanloz, Academic Press, New York, IA, 70, 1969. 22. J . C. I r v i n e and J . C. E a r l , J . Chem. S o c , 121, 2376(1922). 23. Z. E. J o l l e s and W. T. J . Morgan, J . Biochem., 1183(1940). 24. J . C. I r v i n e , D. M c N i c o l l and A. Hynd, 99, 250(1911). 25. M. Bergmann and L. Zervas, Chem. Ber. 64B, 975(1931). 26. M. J . Adam and L. D. H a l l , J . Chem. Soc. Chem. Comm., 234(1979). 27. S. N. Poddar, Z e i t s c h r i f t f u r angorganische und allgemeine Chemie, 322, 326(1963). 28. H. Yo k o i , B u l l . Chem. Soc. Japan, 47, 3037(1974). 29. T. P. Cheesman, D. H a l l and T. N. Waters, J . Chem. Soc. (A), 694 (1966). 30. D. H a l l , R. H. Summer and T. N. Waters, J . Chem. Soc. (A), 420 (1969). 31. R. H. Holm and M. J . O'Connor, Progr, Inorg. Chem., 14, 241(1971). 32. H. P. F r i t z , B. M. G o l l a , H. J . K e l l e r and K. E. Schwarzhans, Z e i t s c h r i f t f u r Naturforschung, 21B, 725(1966). 33. T. J . S w i f t , NMR of Paramagnetic Molecules, eds., G. N. La Mar, W. DeW. Horrocks and R. H. Holm, Academic Press, New York, 1973. 34. C. A l l e y n e , M.Sc. t h e s i s , U n i v e r s i t y of B r i t i s h Columbia, 1973; B. V. McGarvey, T r a n s i t i o n Metal Chemistry, 3^, 89(1966); B. J . Hathaway and D. E. B i l l i n g s , Coordination Chem. Reviews, _5, 143(1970); J . E. Wertz and J . R. B o l t o n , E l e c t r o n Spin Resonance, Elementary Theory and P r a c t i c a l A p p l i c a t i o n s , McGraw-Hill, New York, 1972. CHAPTER I I SYNTHESIS OF SUGAR-ORGANOMETALLIC CONJUGATES: FERROCENYL-MONOSACCHARIDE DERIVATIVES HA: I n t r o d u c t i o n Ferrocene [1] and i t s d e r i v a t i v e s are f i n d i n g 1 an ever i n c r e a s i n g l y wide range of i n d u s t r i a l and biochemical a p p l i c a t i o n s i n areas ranging 2 from the development of new i r o n - c o n t a i n i n g drugs to the formation of 3 . 4 ferrocene-modified polymers and non-toxic "antiknock" f u e l a d d i t i v e s . Such d e r i v a t i v e s are a l s o of i n t e r e s t f o r a number of other reasons i n c l u d i n g the development of o p t i c a l l y a c t i v e c a t a l y s t s ^ of metal com-pounds f o r new immunoassay techniques of heavy metal probes f o r e l e c t r o n 7 8 microscopy , of the synth e s i s of radiopharmaceuticals , and the m o d i f i c a -9 t i o n of e l e c t r o d e surfaces . I t might t h e r e f o r e be a n t i c i p a t e d that these, and oth e r , areas would be advanced i n a v a r i e t y of ways by the development of general routes to the sy n t h e s i s of ferrocenyl-sugar d e r i v a t i v e s . And i t might a l s o be a n t i c i p a t e d that the combined presence 63 Fe IsFcH) 64 of the sugar and metal m o i e t i e s , as suggested i n chapter I , would give these compounds i n t e r e s t i n g b i o l o g i c a l p r o p e r t i e s . Prompted by t h i s , and by the f a c t that carbohydrates can be e i t h e r water s o l u b l e , or when s u i t a b l y s u b s t i t u t e d , s o l u b l e i n organic media, we have i n v e s t i g a t e d the syn t h e s i s of sugar-ferrocene conjugates i n which the sugar moiety i s c o v a l e n t l y attached to the c y c l o p e n t a d i e n y l l i g a n d of the ferrocene com-plex Undoubtedly the most important property of ferrocene i s i t s excep-t i o n a l a r o m a t i c i t y . Ferrocene undergoes a v a r i e t y of t y p i c a l i o n i c aromatic s u b s t i t u t i o n r e a c t i o n s f a r more r e a d i l y than benzene and only the mild e s t r e a c t i o n c o n d i t i o n s are r e q u i r e d . Some of these r e a c t i o n s i n c l u d e F r i e d e l - C r a f t s a c y l a t i o n , a l k y l a t i o n , m e t a l a t i o n , s u l f o n a t i o n and aminomethylation as shown i n Figure I I - l . Ferrocene i s s t a b l e to bases and n o n - o x i d i z i n g a c i d s but i s r e a d i l y o x i d i z e d to the blue f e r r o c i n i u m c a t i o n when t r e a t e d w i t h o x i d i z i n g agents such as n i t r i c a c i d . Ferrocene can be s u b s t i t u t e d on j u s t one r i n g or on both and can possess more than one s u b s t i t u e n t per c y c l o p e n t a d i e n y l r i n g . As w e l l as the i n i t i a l s u b s t i t u t i o n , the products from these r e a c t i o n s can a l s o undergo a v a r i e t y of subsequent r e a c t i o n s to form an immense number of organometallic m a t e r i a l s w i t h a wide range of p r o p e r t i e s . In t h i s chapter, f i r s t the sy n t h e s i s of s u g a r - f e r r o c e n y l complexes w i l l be described. A v a r i e t y of carbohydrates c o n t a i n i n g s u i t a b l e n u c l e o p h i l i c f u n c t i o n a l i t i e s have been reacted w i t h a number of f e r r o -cene d e r i v a t i v e s which are s u s c e p t i b l e to n u c l e o p h i l i c a t t a c k . These r e a c t i o n s have r e s u l t e d i n s e v e r a l organic and aqueous s o l u b l e f e r r o c e n y l -sugar conjugates. Since a l l of these m a t e r i a l s are new organometallic compounds they 65 Figure I I - l . Some t y p i c a l aromatic s u b s t i t u t i o n r e a c t i o n s of ferrocene. 66 have been f u l l y c h a r a c t e r i z e d , as described i n the next s e c t i o n , by nmr spectroscopy. In the f i r s t part of t h i s s e c t i o n , the e f f e c t s of a proximal c h i r a l s u b s t i t u e n t on the proton resonances of the c y c l o -p e n t a d i e n y l r i n g w i l l be examined along w i t h a complete t a b u l a t i o n of a l l chemical s h i f t s and cou p l i n g constants. A l s o , the spec t r a from some of the more i n t e r e s t i n g compounds w i l l be discussed. This i s followed by an account of how proton s p i n l a t t i c e r e l a x a t i o n measurements can be used to a s s i g n the proton resonances of the s u b s t i t u t e d c y c l o p e n t a d i e n y l r i n g and to determine motional c o r r e l a t i o n times. During t h i s s e c t i o n the reader u n f a m i l i a r w i t h t h i s technique, w i l l be r e f e r r e d ahead to the f i n a l s e c t i o n where a more d e t a i l e d e x p l a n a t i o n of the s p i n r e l a x a t i o n e x p e r i -ment i s given. I I B : Synthesis A wide v a r i e t y of ferrocene d e r i v a t i v e s are s u s c e p t i b l e to nucleo-p h i l i c a t t a c k by the amino, hydroxyl or t h i o l f u n c t i o n a l groups of s u i t a b l y blocked carbohydrate d e r i v a t i v e s . A l l of the r e a c t i o n s used here to form f e r r o c e n y l sugar conjugates, are of t h i s type. The com-pounds formed were a l l c r y s t a l l i n e (with exception of [20]) and were u s u a l l y orange or red i n c o l o r . This g r e a t l y f a c i l i t a t e d t h e i r detec-t i o n during t h i n l a y e r chromatography ( t i c ) and as a r e s u l t that tech-nique provided a convenient method f o r f o l l o w i n g r e a c t i o n s ; f o r the same reason i t proved convenient to p u r i f y s e v e r a l compounds by s i l i c a -g e l chromatography (see Experimental s e c t i o n ) . I t was already known that the f e r r o c e n y l c h l o r i d e s [ 2 ] , react r e a d i l y w i t h a v a r i e t y of n u c l e o p h i l i c reagents, i n c l u d i n g t h i o l s , amines, and a l c o h o l s , i n dry organic sol v e n t s w i t h a s u i t a b l e base acceptor and i t was a simple matter to react [2] and [3] w i t h a v a r i e t y 67 8 C CI Fe [ 2 ] [ 3 ] CCl C C l of blocked sugars, each c o n t a i n i n g one of these three f u n c t i o n a l groups. 12 Reaction of 2,3,4,6-tetra-O-acetyl-l-thio-B-D-glucopyranose [4] , and 13 1,3,4,6-tetra-0-acetyl-2-amino-2-deoxy-B-D-glucopyranose [5] occurred r e a d i l y at room temperature i n dry chloroform w i t h e i t h e r p y r i d i n e or OAc AcO AcO OAc t r i e t h y l a m i n e , as the base acceptor, to form the mono and 1,1'-di-t h i o e s t e r s [ 6 ] , [ 7 ] , and amides [ 8 ] , [ 9 ] . Co' ° i © s c A c o ^ n OAc -Fc [6] r-OAc /—°\d A c  N h _ c - F C [61 pOAC NH-C pOftc Fc 1.V NH-C [9] In l i k e f a s h i o n , r e a c t i o n of the mono f e r r o c e n y l c h l o r i d e w i t h 1,2:5,6-di-O-isopropylidene-a-p-glucofuranose [10] i n dry p y r i d i n e overnight af f o r d e d the mono s u b s t i t u t e d e s t e r [11]. 68 14 N,N-Dimethylaminomethylferrocene methiodide [12] a l s o r e a c t s w i t h a v a r i e t y of n u c l e o p h i l i c reagents by v i r t u e of i t s b e n z y l - l i k e s t r u c t u r e and having the t r i m e t h y l amine moiety as a gaseous l e a v i n g group. These Fe Me • I -N- l Me Hr-N-Me 2 i. [12] r e a c t i o n s however, r e q u i r e more vigorous c o n d i t i o n s than those i n v o l v i n g the a c i d c h l o r i d e s . Thus, r e a c t i o n w i t h the 1-thio sugar [4] i n b o i l i n g a c e t o n i t r i l e w i t h anhydrous sodium carbonate was s u c c e s s f u l but a mixture of a and B anomers (40:60) were i s o l a t e d (as determined by *H nmr), probably because of mutarotation of the 1-thio sugar under the b a s i c r e a c t i o n c o n d i t i o n s . F o r t u n a t e l y , s e v e r a l r e c r y s t a l l i z a t i o n s from ethanol afforded the pure B compound [13]. 69 Reaction of the 2-amino sugar [5] w i t h two equiv a l e n t s of the methiodide reagent [12] under the same c o n d i t i o n s y i e l d e d the N,N-bis amine compound [14]. However, t h i s was not a good route to the secondary amine compound [15] s i n c e mixtures (as determined by t i c ) of both [14] and [15] -OAc "ROAc AcO AcO Si [ 1 4 ] N H — C H 2 ~ P c [15] were always obtained even when a la r g e excess of [5] was employed. A l l attempts to couple reagent [12] w i t h sugar hydroxyl groups f a i l e d . The f e r r o c e n y l t o s y l a t e [16] was next evaluated as an a l t e r n a t i v e a l k y l a t i n g reagent to [12]. This compound does not appear to have been synthesized p r e v i o u s l y although i t s intermediacy was pos t u l a t e d i n the formation of the ether [17] during r e a c t i o n of the a l c o h o l [18] w i t h t o s y l c h l o r i d e i n d i e t h y l ether s o l u t i o n 1 " ' . This t o s y l a t e [16] proved to have the same high r e a c t i v i t y as other " b e n z y l i c " t o s y l a t e s and as a CH Fe r O - s o 2 - ^ ^ - M i Fe CHf-0 [ 1 6 ] [ 1 7 ] J 2 CH 20H [18] r e s u l t could not be prepared by the r e a c t i o n of [18] w i t h t o s y l c h l o r i d e i n p y r i d i n e at ambient temperature. Instead i t was prepared by the method of Kochi and Hammond1^ where f i r s t the sodium a l k o x i d e of [18] was prepared w i t h sodium hydride i n ether and then t h i s reacted at low 70 temperature with t o s y l chloride. The tosylate was then used i n s i t u (see Experimental), without i s o l a t i o n . I t should be noted here that the alcohol [18] can be r e a d i l y prepared by reaction of the methiodide [12] with aqueous sodium hydroxide 1^. Reaction of [16] with the 1-thio sugar [4] at ambient temperature for two days gave [13] which was e a s i l y i s o l a t e d since i t c r y s t a l l i z e d r e a d i l y from ethanol. The y i e l d of [13] was much higher than that for the re a c t i o n between [4] and [12] and only the B-anomer was formed. With 1,2:3,4-di-()-isopropylidene-a-D-galactopyranose [19] reaction of [16] i n an ether s o l u t i o n f or two days at room temperature was also success-f u l but the y i e l d was much lower than that found for the thio sugar reac-t i o n . Since t h i s product [20] was an o i l , workup was more d i f f i c u l t and p u r i f i c a t i o n was performed on column chromatography. Reaction of the t o s y l a t e with the 2-amino sugar [5] was also low-yielding and the s o l u t i o n had to be heated under r e f l u x for two days i n a chloro-form/ether s o l u t i o n . The secondary amine product [15] was however, e a s i l y i s o l a t e d since i t could be extracted into d i l u t e acid and then extracted back in t o ether upon n e u t r a l i z a t i o n ; the s t a r t i n g 2-amino sugar was separated from the product since i t was not soluble i n ether. 71 AcO NH-CHj-pC [151 Impressed by the ease w i t h which ^ - t r i a z i n e d e r i v a t i v e s react w i t h 18 carbohydrates (see chapter I I I ) we a l s o evaluated t h i s approach to the sy n t h e s i s of fer r o c e n y l - s u g a r conjugates. Reaction of hydroxymethyl-ferrocene [18] w i t h cyanuric c h l o r i d e [21] i n b a s i c aqueous acetone afforded the f e r r o c e n y l t r i a z i n e d i c h l o r i d e [22]; although the ether [17] was a l s o formed as a byproduct i t could be r e a d i l y removed by CI N ^ N J < ^ ^ > - C H 2 O ^ N ^ \ C I C I ^ N ^ C I Fe [ 2 2 ] f i l t r a t i o n p r i o r to c r y s t a l l i z a t i o n of [22]. The d i c h l o r i d e [22] was somewhat unstable but could be stored f o r s e v e r a l weeks i n an evacuated d e s i c c a t o r and was s u i t a b l e f o r f u r t h e r r e a c t i o n s without p u r i f i c a t i o n . Reaction of [22] w i t h two equivalents of the 1-thio glucose sugar [4] occurred r e a d i l y i n a c e t o n i t r i l e w i t h t r i e t h y l a m i n e to y i e l d the f e r r o -c e nyl sugar compound [23]. This r e a c t i o n w i l l be discussed again i n chapter I I I . 72 pOAc OAc N ^ ^ N AcO OAc [23] Two approaches were considered f o r the formation of water s o l u b l e ferrocene-sugar conjugates: s e l e c t i v e r e a c t i o n of an unblocked sugar and deblocking of the above d e s c r i b e d , blocked d e r i v a t i v e s . I t i s w e l l known that amino sugars form S c h i f f ' s bases w i t h aro-19 matic aldehydes , and r e a c t i o n of ferrocene carboxaldehyde [24] i n ethanol w i t h glucosamine h y d r o c h l o r i d e [25] i n the presence of t r i e t h y l -amine gave, as expected, the S c h i f f ' s base [26] i n 70% y i e l d . This ETOH lETUN'-[ 2 4 ] [25] [26] r e a c t i o n should f i n d s u b s t a n t i a l g e n e r a l i t y . Unfortunately an i n i t i a l attempt to s t a b i l i z e the S c h i f f ' s base by r e d u c t i v e amination w i t h sodium cyanoborohydride was uns u c c e s s f u l ; t i c of the r e a c t i o n mixture i n d i c a t e d the formation of a mixture of compounds. I t should be noted here that 20 the aldehyde [24] can be prepared from the methiodide [12] Attempts to deblock the s u b s t i t u t e d sugar conjugates were very d i s a p p o i n t i n g s i n c e only two r e a c t i o n s gave acceptable y i e l d s . Thus de-O-acetylation of compounds [6] and [13] was s u c c e s s f u l l y accomplished >Ac o • ° v S - ? - F c 21 using sodium methoxide i n methanol • Compound [13] was c l e a n l y deacetylated and the product [27] c r y s t a l l i z e d r e a d i l y from water as the monohydrate. Compound [ 6 ] , as expected under these c o n d i t i o n s , was p a r t i a l l y cleaved at the t h i o - e s t e r l i n k a g e but the main product was the unblocked f e r r o c e n y l d e r i v a t i v e [28]; the product was e a s i l y p u r i f i e d by column chromatography and the methyl ferrocenecarboxylate was a l s o i s o l a t e d , and subsequently i d e n t i f i e d by *H nmr. D e a c e t y l a t i o n of the l , l ' - d i - t h i o e s t e r [7] proved to be very complex ( t i c ) and was not pur-sued f u r t h e r . More s u r p r i s i n g l y , d e a c e t y l a t i o n of both the amides [8] and [9] w i t h sodium methoxide i n methanol was complex and no pure product could be i s o l a t e d . D e a c e t y l a t i o n of the amines [14] and [15] a l s o proved d i s a p p o i n t i n g ; i n each case two compounds were detected by t i c , which had very s i m i l a r values and attempts to p u r i f y these compounds by p r e p a r a t i v e t i c or by column chromatography f a i l e d . The ^ - t r i a z i n e d e r i v a t i v e [23] was a l s o exposed to these d e a c e t y l a t i o n c o n d i t i o n s but t i c showed the major product to be c o l o r l e s s ( d e t e c t i o n by l^SO^ c h a r r i n g ) , 74 which suggests cleavage of e i t h e r the t h i o sugar or ferrocene moieties from the t r i a z i n e r i n g . Removal of the i s o p r o p y l i d e n e b l o c k i n g groups from [20] was a l s o u n s u c c e s s f u l . The standard procedure of h e a t i n g i n d i l u t e s u l f u r i c a c i d could not be used s i n c e ferrocene compounds are r e a d i l y o x i d i z e d under these c o n d i t i o n s . Heating under r e f l u x i n aqueous methanol using IR 120 H + r e s i n was t r i e d but a f t e r 1 h the s o l u t i o n turned c o l o r l e s s and a c o l o r l e s s product was detected on t i c having the same R^ value as the diacetone galactose precursor [19], suggesting that cleavage of the ether l i n k a g e was t a k i n g place. The r e s i n had turned green a l s o and i t was thought that o x i d a t i o n was o c c u r r i n g under these c o n d i t i o n s as w e l l . I I C: Proton Nuclear Magnetic Resonance Spectra ( i ) Chemical S h i f t s and Coupling Constants The *H chemical s h i f t s and coupling constants f o r the f e r r o c e n y l sugar d e r i v a t i v e s [ 6 ] , [ 7 ] , [ 8 ] , [ 9 ] , [11], [13], [14], [15], [20], [23], [26], [27], [28] are summarized i n Table I I - 1 . I t i s i n t e r e s t i n g to observe that a proximal c h i r a l group renders a l l four protons of the s u b s t i t u t e d c y c l o p e n t a d i e n y l r i n g i n e q u i v a l e n t , 22 as p r e v i o u s l y observed by Kursanov f o r cymantrene systems. The c h i r a l group, i n our case the sugar, renders the a, a' and 6, B' protons R Fe R' TABLE 1. C h e m i c a l S h i f t s (ppm) and m u l t i p l e ! s p l i t t i n g s (Hz) f o r t h e f e r r o c e n y l m o n o s a c c h a r i d e compounds Compound ( a > a ) a a (Unsub) H-1 H-2 H-3 H-A H-5 H-6 H-6' OAc - C H 2 6 b 4.77 3.99 3.97 5.72 5.54 5.48 5.32 3.34 4.22 4.0 1.70 4.68 J[ 210.4 J 2 3 10.4 J 3 j l ) 9 . 0 J,, 5 10.0 J 5 > 6'2.0 1.69 1.68 1.65 b 7 4.76 4.11 5.70 Ca.5.5 tYl.5.5 5.35 3.50 4.29 4.05 1.79 4.61 4.02 J, 2 10.5 J ^ > 5 10.3 J 5 > 6 *-3 J 6 >6'12.6 J 5 ) 6'2.0 1.71 v 2 ' 6 1.69 1.65 b c 8 4.71 4.01 4.05 5.90 4.76 5.42 5.35 3.64 4.31 4.13 1.73 4.68 J , 2 8.8 J 2 3 10.0 J 3 > „ 9.4 J ^ 5 9.4 J 5 6 < 2.1 1.70 1.67 1.61 9 d 6 Ca.4.3 Ca 4.3 5.99 4.48 5.43 5.23 3.94 4.35 4.16 2.14 J[ 2 8.8 J 2 3 10.1 J 3 > ^ 9 . 5 Ji, 5 9.8 J 5_ 64.4 J 6 6 ' 1 2 . 4 J 5 > 6 ' 2.1 2.12 2.10 2.09 VI TABLE 1. C o n t i n u e d a a Compound ( a . a ) (B>3) C P H - l H-2 H-3 H-A H-5 H-6 H-6' OAc - C H 2 (Unsub) l l b 1 4.97 4.07 4.10 5.73 4.36 5.78 4.51 4.51 4.11 4.24 J , 2 3-6 J 3 i „ 2.0 J „ 5 2 . 3 J 5 6 5.3 J 6 j 6 ' 8 . 7 J 5 6 ' 3.5 13 b 4.15 3.93 3.99 5.37 4.23 5.37 5.25 3.24 4.25 A.06 1.73 3.72 J 5 6 4.9 Jf,y 12.3 j 5 6/ 2.2 1.72 3.59 1.69 1.67 Jgem 12.7 14 b 4.23 3.96 4.03 5.99 3.34 5.59 5.26 3.25 4.28 4.0 1.80 3.90 Jj 2 8-9 J2 3IO.6 J 3 1, 9.1 J M 5 10.0 J 5 6 4.1 J 5 6 ' 1.9 1.70 3.70 1.68 Jgem 13.2 1.65 ^ 4.11 3.93 4.02 5.72 2.98 5.20 5.32 3.32 4.31 4.01 1.75 3.64 J, 2 8.5 J 2 3 10.0 J 3 „ 9.8 J,, 5 9.3 J 5 6 4. 1 J 6 > 6 ' 12.4 j $ 6' 2.2 1.70 3.46 1.66 Jgem 13.2 1.63 b 0 2 0 3.98 5. 55 4.17 4.51 TABLE 1. C o n t i n u e d a a Compound ( a , a ' ) (b»b') c P H-1 H-2 H-3 H-4 H-5 H - 6 H-6' OAc " C H 2 (Unsub) 2 3 b H 4.59 3.87 3.92 H-l x5.83 H-2, 5,44 H-3„5.34 H-4,5.26 H-5^3.26 4.16-4.18 4.03-4.08 1.86 4.83 J U 2,10. 5 J'., 5,10.2 J5.6.5.2 •»i,6« 2.0 1.74 4.33 H-l r5.44 H-2y 5.34 H-3j 5.34 11-4^5.16 H-5y 3.34 1.71 4.09 J, 2 10.5 J*,, 5,10.1 J5,6,4.2 1.69 4.16-4.18 4.03-4-08 1.68 \4 2-1 1.67 Jgem 15.0 2 J i h 4.47 4.24 H-1,5.86 H-2, 5.25 H-3, 5.54 H-4 X5.25 2.03 5.05 4.18 J l , 2 , 10.6 J 2 , 3,9.3 J 3, 1,9.3 ca.k . 1 ca.4.1 ca.4.1 2.02 4.40 H-ly 5.76 H-2^5.11 H-3y5.47 H-4y5.08 2.02 4.81 J l 2.10.6 2.01 1.99 Jgem 15.1 1.98 1.97 1.97 1 k 26 4.27 2.88 3.61 3.70 3.91 J , 2 10-2 J 2 3 ' - I J 3 > 1 , 9.2 J 5 | 6 5.2 J 6 6 ' 11.6 J 5 6 ' 1.7 27 1 4.27 4.12 4.17 4,37 3.27 3.4 3.4 •v 3.4 3.69 3.89 3.84 J l 2 9-6 J 2 3 9.4 J 5 65.4 J 6 6'12.2 J5 6'2. 2 3.71 Jgem 13.2 TABLE 1. C o n t i n u e d Compound a a (a,a ) ( g - B ) Cp (Unsub) H-1 H-2 H-3 H-4 H-5 H-6 H-6' OAc - C H 2 J 28 4.83 4.49 4.34 4.76 4.87 3.69 3.52 3.46 3.70 3.89 a assignments were made by comparison to 6 and 7 (22); a and 0 resonances are assigned to high f i e l d , of t h e i r a and 8 counterparts b Deuteriobenzene s o l u t i o n 0 1 Deuterioacetone s o l u t i o n • I " c N-C- 5.89, JNH.Hj - 9.1 Hz H j Di, Methanol s o l u t i o n . k I d Deuterlochloroform s o l u t i o n N = CH 8 15 I 0 1 Deuterioacetone/D 20, ca. 10:1 s o l u t i o n . I II e N-C- 6.90, JNH.Hj- 9.3 Hz H f o Me 1.39, 1.32, 1.23, 1.04 M h Since there are two l n e q u i v a l e n t sugar r i n g s the n o t a t i o n H-ljf^H-ly e t c . i s used. o Me g o Me 1.50, 1.42, 1.16, 1.04 o ' ^ M e 79 d i a s t e r e o p t o p i c and p o t e n t i a l l y four separate resonances can be observed. This turned out to be the case i n the spectrum of compound [7] (Figure I I - 2 ) , but more u s u a l l y only the a protons are separated and the 8 pro-tons remain overlapping as i n the spec t r a of compounds [6] and [8] i n Figure I I - 3 and Figure II-4 r e s p e c t i v e l y . I t should be noted that 1 3C 23 chemical s h i f t s are more s e n s i t i v e to t h i s e f f e c t The spectrum of compound [7] i n Figure I I - 2 r e v e a l s the i n e q u i v a l -ence of the four c y c l o p e n t a d i e n y l (Cp) r i n g protons and ^H-1!! s p i n decoupling can r e a d i l y be used, as shown, to assign which protons are on the same s i d e of the r i n g , that i s the p a i r i n g of a w i t h 3 and a' w i t h B^. This spectrum a l s o shows a f a i r l y normal p a t t e r n f o r the sugar r i n g protons except that the overlap of resonances H 2 and H3 r e s u l t i n some second-order e f f e c t s , observed i n the form of " e x t r a " t r a n s i t i o n s i n a d d i t i o n to those expected on a f i r s t - o r d e r b a s i s . The spec t r a of compounds [6] and [8] i n Figures I I - 3 , 4, as was s t a t e d , both show the more usual p a t t e r n of the s u b s t i t u t e d Cp r i n g protons, where the a and a' resonances are separated, and the 8 and 8 ' resonances are overlapping. The unsubs t i t u t e d Cp r i n g appears as a sharp s i n g l e t at about 4 ppm. The resonances of the sugar r i n g protons i n the spectrum of [8] f i t the normal p a t t e r n whereas i n the spectrum of [7 ] , " v i r t u a l c o u p l i n g " i s again evident. The decoupling experiment i n Figure I I - 3 f o r compound [6] r e v e a l s the very small a-a' cou p l i n g when the 8 resonances are i r r a d i a t e d . I t should be pointed out here that the b e a u t i f u l d i s p e r s i o n of a l l four Cp r i n g protons i n the spectrum of [7] was only obtained i n Deuteriobenzene; i n c o n t r a s t , i n other s o l v e n t s such as CDC&3 the 8 protons were overlapping. While the spec t r a of most of the compounds i n Table I I - l i l l u s t r a t e nothing new from what has j u s t 80 Figure I I - 2 . Proton nmr s p e c t r a (270 MHz) of compound [7] i n d e u t e r i o -benzene. The upper t r a c e i s a decoupling experiment used to assign a, a' and B, B' c y c l o p e n t a d i e n y l resonances. 81 Figure I I - 3 . Proton nmr spe c t r a (270 MHz) of compound [6] i n d e u t e r i o -benzene. The upper trace i s a decoupling experiment showing the a-ct' co u p l i n g (^  1 Hz), when 3, 3' i s i r r a d i a t e d . Figure I I - 4 . Proton nmr spectrum (270 MHz) of compound [8] i n deuteriobenzene. oo 83 been discussed, the *H spectrum i n Figure I I - 5 of compound [23] i s , how-ever, very i n t e r e s t i n g indeed and merits separate d i s c u s s i o n . The proton resonances of the two sugar r i n g s i n t h i s compound are non equiv-a l e n t i n the *H nmr spectrum which i m p l i e s that the molecule must favour a p r e f e r r e d set of conformations. Although there e x i s t s no formal sym-metry, elements i n t h i s molecule, i t was argued i n i t i a l l y that a pseudo-C 2 r o t a t i o n a x i s , as shown below, could be generated i f there were free r o t a t i o n ( f a s t on the nmr time s c a l e ) about the bonds j o i n i n g the s u b s t i t -uents to the c e n t r a l t r i a z i n e r i n g . However, the observed non e q u i v a l -ences imply that s t e r i c hindrance prevents complete r o t a t i o n about some of these bonds. In the i n s e r t i n Figure I I - 5 , of the acetate r e g i o n , a l l e i g h t acetate groups are r e s o l v e d . Since the separation between two acetate resonances, common to both sugar r i n g s , could not be more than 15 Hz, i t would t h e r e f o r e be necessary f o r the bond to r o t a t e slower than 2tt x 15 r e v o l u t i o n s per second i n order f o r separate resonances to be observed. T h i s , however, would correspond to an u n r e a l i s t i c a l l y high b a r r i e r to r o t a t i o n ; i t f o l l o w s that our o r i g i n a l argument i s untenable. The f o l l o w i n g e x p l a n a t i o n appears to be p r e f e r a b l e . I t seems l i k e l y t h at the molecule favours a conformation i n which the ferrocene moiety i s o r i e n t e d on one s i d e of the t r i a z i n e r i n g as shown i n the f o l l o w i n g diagram. This would a l l o w a r a p i d " cranking" motion about the bonds I 1 1 6 5 8 (ppm) 4 Figure I I - 5 . Proton nmr s p e c t r a (270 MHz) of compound [23] i n deuterioacetone. The top l e f t i n s e r t shows the H c and H r resonances of [23] i n deuteriobenzene 5x 5y and the top r i g h t i n s e r t shows the eight acetate resonances of [23] i n deuterioacetone. 85 < 0 ? Fe j o i n i n g the ferrocene group to the t r i a z i n e moiety which would be reason-able since t h i s motion sweeps out only a small volume. I t i s then assumed that the bond r o t a t i o n s which would a l l o w the ferrocene u n i t to t i p over to the underside of the r i n g must be very slow. This seems al s o to be reasonable since a la r g e i n e r t i a l moment must be overcome i n order f o r the bulky ferrocene group to make a 360° sweep through solvent molecules; an "absence" of that motion e l i m i n a t e s any symmetry f o r the molecule. I t can then be seen from molecular models that the p r e f e r r e d conformation f o r the sugar u n i t s places one sugar u n i t above and one below the plane of the t r i a z i n e r i n g as shown i n the above diagram. These two e f f e c t s combined would then b r i n g one sugar group much c l o s e r to the aromatic r i n g current of the ferrocene system than the other sugar group and r e s u l t i n a chemical s h i f t d i f f e r e n c e between resonances of the two sugar m o i e t i e s . This argument i s supported by the spectrum i n Figure I I - 5 , s i n c e the sugar r i n g resonances Hi+Hi, f o r one sugar group, 86 are a l l s h i f t e d i n one d i r e c t i o n w i t h respect to the equivalent reson-ances of the other sugar group. That i s , H^^ i s to low f i e l d of H-^y> i s to low f i e l d of e t c - The spectrum of t h i s compound should be compared to the s i m i l a r s p i n l a b e l compound ([36] chapter H I D ) . Both sugar r i n g s appear equivalent i n t h i s compound because the n i t r o x i d e u n i t i s much l e s s bulky than the ferrocene moiety, a l l o w i n g i t to r o t a t e more f r e e l y , and a l s o i t does not impart the same, l a r g e chemical s h i f t p e r t u r b a t i o n to the system as does the aromatic ferrocene group. An i n t e r e s t i n g feature of the deuteriobenzene spectrum of [23] i s that the H5 resonances, shown i n the i n s e r t of Figure I I - 5 , of the two sugar r i n g s , e x h i b i t q u i t e d i f f e r e n t c o u p l i n g p a t t e r n s . The H,. reson-ance has a much l a r g e r Jr , =5.2 Hz than does the more usual value f o r 5y,6y H,. of J , , = 4.2 Hz. This suggests that the bond j o i n i n g C_ and 5x 5x,6x 5y C, , as shown below, i s fa v o u r i n g p r e f e r r e d conformations, which 6y r e i n f o r c e s the previous suggestion that t h i s molecule i s s t e r i c a l l y crowded. ( i i ) Proton Spin L a t t i c e R e l a x a t i o n Rates The *H nmr spectrum of an a c h i r a l , monosubstituted ferrocene d e r i v a t i v e i n v a r i a b l y shows two m u l t i p l e t s , each from a p a i r of equi a l e n t protons {a,a' and B,B'}, together w i t h a sharp s i n g l e t f o r the f i v e e q u i v a l e n t protons of the un s u b s t i t u t e d r i n g . When both r i n g s 87 s u b s t i t u t e d by the same a c h i r a l s u b s t i t u e n t , only two resonances are observed, both r i n g s being e s s e n t i a l l y i d e n t i c a l . And as mentioned i n the l a s t s e c t i o n f o r c h i r a l , mono or l , l ' - d i - s u b s t i t u t e d d e r i v a t i v e s , a l l four proton resonances of these r i n g s are i n t r i n s i c a l l y i n e q u i v a l e n t . Although i n some cases a reasonable assignment of such resonances can be i n f e r r e d by i n s p e c t i o n , f o r many systems not even a t e n t a t i v e assignment can be made; i t w i l l now be shown that proton s p i n l a t t i c e r e l a x a t i o n r a t e s (Ri-values) provide d i r e c t evidence f o r unequivocal assignments. As an added bonus those same data a u t o m a t i c a l l y provide i n f o r m a t i o n concerning the r e l a t i v e m o b i l i t i e s of the s u b s t i t u t e d and u n s u b s t i t u t e d c y c l o p e n t a d i e n y l r i n g s ^ 4 . Although to our knowledge, no r e s u l t s have p r e v i o u s l y been reported f o r organometallic fr-complexes, an ample body of data e x i s t s f o r organic molecules which shows that the s p i n l a t t i c e r e l a x a t i o n of the protons of most diamagnetic organic molecules i s dominated, g e n e r a l l y e x c l u s i v e l y , 25 by the d i p o l e - d i p o l e mechanism , which has the general form 2 2 R!(D,R) - D R • x (D+R) (1) r 6(r»R) C where R!(D,R) i s the s p e c i f i c r e l a x a t i o n c o n t r i b u t i o n between a donor nucleus (D) and a receptor nucleus (R), y and y d a r e t n e gyromagnetic r a t i o s of those two n u c l i d e s , r(IH-R) i s the di s t a n c e separating them and t^CIH-R) i s the motional c o r r e l a t i o n time of the vector j o i n i n g D and R. For most diamagnetic molecules protons are the only nuclear species w i t h a high gyromagnetic r a t i o and f o r that reason the r e l a x a t i o n of each proton g e n e r a l l y occurs v i a the other protons of the system; working at high d i l u t i o n i n a solvent which contains no protons ensures that i n t r a -molecular proton r e l a x a t i o n i s dominant. Under these circumstances i t 88 i s p o s s i b l e to use the experimentally determined values of RjCD.R) to measure e i t h e r the r e l a t i v e i n t e r p r o t o n d i s t a n c e s or the r e l a t i v e r a t e s of motion of the va r i o u s proton-containing m o i e t i e s . In the context of ferrocene chemistry, two u s e f u l items of informa-t i o n can be obtained from a simple, q u a l i t a t i v e i n t e r p r e t a t i o n of proton R l values. Because protons which are a to a s u b s t i t u e n t have only one neighbouring proton whereas those which are 3 have two, the l a t t e r w i l l be c h a r a c t e r i z e d by t h e i r l a r g e r R} values. And, i f i t i s assumed that a l l C-H bond lengths and bond-angles are i d e n t i c a l , intercomparison of the Rj values can show which c y c l o p e n t a d i e n y l r i n g i s r o t a t i n g about the Cp-Fe-Cp a x i s more r a p i d l y . For b r e v i t y we s h a l l i l l u s t r a t e these p o i n t s here using the mono- and b i s - s u b s t i t u t e d sugar d e r i v a t i v e s [6] and [7]. 0 [6] R = - C - S G l c , R = H 0 [7 J : R = R'= - C - S G L c i-OAc HSGLc = ((0AcV A c 0 OAc The 270 MHz proton resonance s p e c t r a of compounds [6] and [7] are shown i n Figure I I - 6 and the r e l e v a n t proton Ri values are summarized i Table I I - 2 . I t i s c l e a r that although the R e v a l u e s of the resonances at 4.77 ppm and 4.68 ppm of [6] are c l o s e l y s i m i l a r , both these are r e l a x i n g at approximately h a l f the r a t e of the resonances at 3.99 ppm. C l e a r l y the l a t t e r resonances must correspond to B, g', t h e i r enhanced 89 Figure H-6. P a r t i a l proton nmr spectra of (A) compound [6] and (B) compound [ 7 ] , i n deuteriobenzene. 90 TABLE I I - 2 . Proton s p i n l a t t i c e r e l a x a t i o n r a t e s Rj sec 1 Cp a, a" 8, 3 ' (unsub) 0 II [6] R=-C-SGlc, R'=H 0.258 0.426 0.185 0.280 0 [7] R=R'=-C-SGlc 0.483 0.84* 0.482 0.780 *Due to an a c c i d e n t a l overlap w i t h a sugar resonance t h i s r a t e was estimated from n u l l p oint determinations. R^-values being a s c r i b e d to the f a c t that they each have two neighbouring protons. A s i m i l a r r e l a t i o n s h i p p e r t a i n s f o r [ 7 ] , the i n t e r p r e t a t i o n being only m a r g i n a l l y complicated by the a c c i d e n t a l overlap between the 3 resonance and one of the protons of the sugar r i n g . Assignment of which protons are on the same side of the cyclopenta-d i e n y l r i n g , that i s the p a i r i n g of a w i t h 8 and a' w i t h 8 ' i n I I , i s t r i v i a l l y accomplished by homonuclear decoupling as was shown i n Figure I I - 2 . U n f o r t u n a t e l y , i t i s not easy to u l t i m a t e l y a s s i g n each resonance to a p a r t i c u l a r proton and we can t h i n k of no simple method whereby t h i s can be accomplished. Although not d i r e c t l y r e l a t e d to the main t h r u s t of the work presented thus f a r i n t h i s s e c t i o n , I would l i k e to i l l u s t r a t e to the reader not 25 f a m i l i a r w i t h t h i s technique, the conformational i n f o r m a t i o n obtainable from r e l a x a t i o n r a t e s by examining the R e v a l u e s f o r protons of the append-ed sugar r i n g i n (6) (Figure I I - 7 ) . As shown e a r l i e r i n equation [ 1 ] , the r e l a x a t i o n r a t e i s dependent upon the distance between protons and because 91 3 0.54 Figure I I - 7 . Conformational s t r u c t u r e and proton Rj^-values f o r the appended sugar moiety i n compound [6]. of the r ^ dependence the r e l a x a t i o n c o n t r i b u t i o n s drop o f f sharply as the distance i s increased. Due to t h i s , the v i c i n a l t r a n s - d i a x i a l i n t e r a c -t i o n s , present between neighbouring protons i n t h i s p a r t i c u l a r sugar r i n g , are s m a l l and c o n t r i b u t e l i t t l e to the R e v a l u e s of the r i n g protons. The R^-value f o r Hg i s the l a r g e s t observed because there i s very e f f i c i e n t r e l a x a t i o n between the c l o s e l y spaced geminal protons and there i s a l a r g e c o n t r i b u t i o n from H5 as w e l l . The Rj-value of H5 i s the next l a r g e s t since i t gets r e l a x a t i o n c o n t r i b u t i o n s from both Hg protons and the two s y n - d i a x i a l protons Hi and H3. The Rj-values f o r protons Hi+ and H} provide an i n t e r e s t i n g comparison. Since H^ has 1 , 3 - d i a x i a l i n t e r a c -t i o n s w i t h both H3 and H5, i t r e l a x e s more r a p i d l y than Hi+ which has only the d i a x i a l i n t e r a c t i o n w i t h H 2. As expected, the R e v a l u e of H 2 i s the sm a l l e s t s i n c e i t gets r e l a x a t i o n only from while Hi+ gets some 92 a d d i t i o n a l r e l a x a t i o n from the Hg protons. The r a t e f o r H3 seems a l i t t l e low since i t has two 1 , 3 - d i a x i a l i n t e r a c t i o n s w i t h H5 and but i t i s s t i l l w i t h i n reason and, sin c e there may w e l l be a l a r g e r systematic e r r o r i n the Rj-values of H2 and H3 because they are p a r t i a l l y o v erlapping and 2 6 s t r o n g l y coupled , i t i s not worth d i s c u s s i n g t h i s point f u r t h e r . I t should be c l e a r from the above that conformational i n f o r m a t i o n can be r e a d i l y obtained v i a the r e l a x a t i o n technique f o r ferrocene and other c l a s s e s of organometallic compounds as w e l l . Information concerning the r e l a t i v e r a t e s of spi n n i n g motion of the s u b s t i t u t e d and unsubs t i t u t e d c y c l o p e n t a d i e n y l r i n g s about the Cp-Fe-Cp a x i s can be i n f e r r e d d i r e c t l y from the proton Rj-values. Simply, the observation that the 8 protons of the s u b s t i t u t e d r i n g r e l a x f a s t e r than the protons of the uns u b s t i t u t e d r i n g immediately i m p l i e s that the l a t t e r i s r o t a t i n g f a s t e r than the former. A value f o r t h i s r a t e d i f f e r e n c e can be approximated i n the f o l l o w i n g way. Using equation (2) the r e l a x a t i o n R!(R) = I Ep (D,R) (2) c o n t r i b u t i o n p(D,R) of the donor nucleus to the rec e p t o r , can be c a l c u l a t e d from the observed Rj-value of the receptor nucleus. A f t e r f i r s t c a l c u l a t -i n g p ( a , 8 ) = 0.18 t h i s can then be used to c a l c u l a t e a value f o r p ( 8 , 8 ) = 0.11; and p(H,H)=0.62 can be c a l c u l a t e d s e p a r a t e l y f o r the unsu b s t i t u t e d c y c l o p e n t a d i e n y l r i n g . From the r e l a t i o n s h i p Y ^ J h 2 p(D,R) = 4/3 I p d p + D x (D-^ -R) (3) R R r6(l»R) ° where I i s the nuclear s p i n and other terms are as f o r (1) , one can c a l -c u l a t e the motional c o r r e l a t i o n time f o r the two i n t e r p r o t o n v e c t o r s a+8(x =1.22xl0" 1 0 sec/rad.) and B+8(T =0.71><10~10 s e c / r a d . ) , using a c c 93 o value of the i n t e r p r o t o n distance r = 2.7A; t h i s d i s t a n c e need not be accurate s i n c e only r e l a t i v e r a t e s are being c a l c u l a t e d . An average value f o r the c o r r e l a t i o n time of the i n t e r p r o t o n v e c t o r s f o r the unsub-s t i t u t e d r i n g i s c a l c u l a t e d from p(H,H) to be 0.42 x 10 sec/rad. By comparing the average value of xc(a-»-8) and T (8-*-8) f o r the s u b s t i t u t e d r i n g w i t h the average x^ value obtained f o r the u n s u b s t i t u t e d r i n g i t i s c l e a r that the u n s u b s t i t u t e d r i n g i s r o t a t i n g about" the Cp-Fe-Cp a x i s approximately 2.3 times more r a p i d l y than the s u b s t i t u t e d r i n g . In t h i s c a l c u l a t i o n i t has been assumed that other molecular motions of the molecules c o n t r i b u t i n g to the r e l a x a t i o n r a t e s of these r i n g protons are approximately the same f o r both r i n g s , and t h e r e f o r e e f f e c t i v e l y c a n c e l , l e a v i n g the d i f f e r e n c e i n these r a t e s a f f e c t e d mainly by t h e i r r o t a t i o n a l r a t e s about the Cp-Fe-Cp a x i s . This r a t e d i f f e r e n c e i s i n t u i t i v e l y reasonable and the d i f f e r e n t i a l can be ascribed to the increased s i z e and i n e r t i a l moment of the s u b s t i t u t e d r i n g . I t i s a l s o worth n o t i n g that a two-factor d i f f e r e n t i a l e x i s t s between the R^-values f o r the a protons of [6] and [7] and f o r the B counterparts. Once again, the sense of t h i s d i f f e r e n t i a l i n d i c a t e s that the l a r g e r molecule [7] i s tumbling more slowly o v e r a l l than i t s smaller counterpart [ 6 ] ; t h i s probably r e f l e c t s the increased drag a s s o c i a t e d w i t h the second sugar s u b s t i t u e n t . The r e l a x a t i o n r a t e s obtained f o r Table II - 2 were a l s o checked by roughly e s t i m a t i n g the r a t e s from the n u l l p o i n t s of i n d i v i d u a l resonances i n the p a r t i a l l y r e l a x e d s p e c t r a using the r e l a t i o n s h i p R: = ^ (4) where t i s a short delay a f t e r the 180° pulse to a l l o w the nuclear s p i n to p a r t i a l l y r e l a x . 94 Two concluding statements seem to be appropriate. F i r s t , that the proton Rj values of many d i f f e r e n t c l a s s e s of organometallic substances are l i k e l y to be amenable to the same simple, u s e f u l i n t e r p r e t a t i o n s as those given here. Second, that s i n c e i t i s p o s s i b l e to extend the r e l a x a t i o n experiment to a q u a n t i t a t i v e measurement of i n t e r p r o t o n d i s -27 tances w i t h an accuracy which under favourable c o n d i t i o n s can approach that of a neutron d i f f r a c t i o n study, use of t h i s technique to i d e n t i f y the p o s i t i o n s of the hydride s u b s t i t u e n t s of c e r t a i n metal-hydrides c l e a r l y m e r its a t t e n t i o n . IID: Proton S p i n - L a t t i c e R e l a x a t i o n The f o l l o w i n g s e c t i o n has been adapted i n part from a Ph.D. t h e s i s 25 26 by Dr. K. F. Wong, and s e v e r a l recent reviews on the subject ' Con v e n t i o n a l l y , three sets of nmr parameters: chemical s h i f t s , c o u p l i n g constants, and i n t e g r a t e d areas, are used as the b a s i s f o r s t r u c -t u r a l assignment. T h i s , however, negl e c t s two f u r t h e r sets of magnetic resonance parameters; these are s p i n - l a t t i c e r e l a x a t i o n r a t e s (Rj-values) and the s p i n - s p i n r e l a x a t i o n r a t e s . Recent instrum e n t a l developments have made p o s s i b l e the r o u t i n e measurement of s p i n - l a t t i c e r e l a x a t i o n r a t e s , and t h i s technique i s now f i n d i n g many a p p l i c a t i o n s i n organic and . 25,26,27 in o r g a n i c chemistry Studies of s p i n - l a t t i c e r e l a x a t i o n are b a s i c a l l y concerned w i t h the way i n which, and the rate at which, magnetic energy i s t r a n s f e r r e d between the magnetic n u c l e i under study (the "spins") and t h e i r surround-i n g environment (the " l a t t i c e " ) . This energy i s t r a n s f e r r e d by i n t e r -a c t i o n s between a r a p i d l y f l u c t u a t i n g magnetic f i e l d , generated and loca t e d i n the l a t t i c e , and the r a p i d l y processing n u c l e i of i n t e r e s t . 95 There are a number of d i s t i n c t mechanisms whereby t h i s energy t r a n s f e r can be e f f e c t e d ; f o r t u n a t e l y , however, i t i s o f t e n p o s s i b l e to conduct experiments i n which one of these mechanisms, the i n t r a m o l e c u l a r d i p o l e -d i p o l e mechanism, dominates the r e l a x a t i o n . In p r a c t i c e , t h i s r e q u i r e s that one be i n t e r e s t e d i n molecules which are moving more or l e s s i s o -t r o p i c a l l y i n s o l u t i o n . Furthermore, these molecules should be studied as a d i l u t e s o l u t i o n (ca. < 0.1 M) i n a magnetically i n e r t s o l v e n t , that i s , a solvent that contains n e i t h e r f l u o r i n e nor proton atoms; f o r example, a deuterated organic s o l v e n t . The mathematical form of the i n t r a m o l e c u l a r d i p o l e - d i p o l e mechanism was shown p r e v i o u s l y i n H C ( i i ) and i s given here again i n abbreviated form i n (1) where Rj(D,R) i s the s p e c i f i c r e l a x a t i o n c o n t r i b u t i o n between a donor nucleus (D) and a receptor nucleus (R), y and y are the gyromagnetic r a t i o s of those two n u c l i d e s , r(r»R) i s the d i s t a n c e s e p a r a t i n g them, and T (D->R) i s the motional c o r r e l a t i o n time of the v e c t o r j o i n i n g D and R. I f the c o n d i t i o n s can be met to ensure a dominance of the d i p o l e - d i p o l e mechanism, which i s u s u a l l y obtainable i f one has access to a FT s p e c t r o -meter, then i t can be seen, from equation (1) that both " d i s t a n c e " informa-t i o n and "motional" i n f o r m a t i o n ( c o r r e l a t i o n times) can be e x t r a c t e d from s p i n l a t t i c e r e l a x a t i o n r a t e s . For most diamagnetic molecules, protons are the only nuclear species w i t h a high gyromagnetic r a t i o and f o r that reason the r e l a x a t i o n of each proton g e n e r a l l y occurs v i a the other protons w i t h i n the same molecule. Since the r e l a x a t i o n r a t e Rj_ f a l l s o f f as the.inverse of the s i x t h power of the i n t e r n u c l e a r s e p a r a t i o n of D and Rl(D,R) -6(EH-R) • x (D+R) c (1) 96 R then each proton r e c e i v e s most of i t s r e l a x a t i o n from i t s nearest neighbour protons. Of course, i n most " r e a l " molecules, each i n d i v i d u a l proton w i l l be r e l a x e d by i n t e r a c t i o n s w i t h s e v e r a l other protons ( D - l , D-2, . . .) i n the same molecule, p r o v i d i n g they are c l o s e enough, and the t o t a l r e l a x a t i o n r a t e R\(R) w i l l have the form given i n equation ( 5 ) . Rl (R) = RiCD-l) + R x(D-2) + . . . (5) This summation can be s t a t e d i n another way as shown i n equation (2) R X(R) = 3/2 Zp(D,R) (2) where p(D,R) i s the r e l a x a t i o n c o n t r i b u t i o n of the i n d i v i d u a l donor n u c l e i to the r e c e p t o r , and has the form, R R r 6(D+R) C p(D,R) = 4/3 I.(I B+1) x (D-^ R) (3) where I i s the nuclear s p i n and other terms are as f o r equation (1). Therefore, i f i s known then i t may be p o s s i b l e to c a l c u l a t e these i n d i v i d u a l r e l a x a t i o n c o n t r i b u t i o n s and hence c a l c u l a t e i n d i v i d u a l proton-proton d i s t a n c e s and c o r r e l a t i o n times f o r the v e c t o r s connecting these protons, as was done i n s e c t i o n H C ( i i ) . The simplest conceptual model, i n v o l v i n g pulse-nmr methods, upon which the b a s i c Spin L a t t i c e R e l a x a t i o n experiment i s based, i s the " r o t a t i n g - r e f e r e n c e frame" model. An ensemble of m a g n e t i c a l l y equivalent n u c l e i (spins) subject to the i n f l u e n c e of an e x t e r n a l magnetic f i e l d BQ, at thermal e q u i l i b r i u m between the spins and the l a t t i c e , w i l l have a net, macroscopic magnetic moment which w i l l be d i r e c t e d along the z - a x i s , i n the r o t a t i n g reference frame, along w i t h BQ (see Figure I I - 8 ) . In Figure I I - 8 t h i s magnetic moment i s represented by a v e c t o r , whose length i s equivalent to the t o t a l amount of magnetization present. However, when the vector l i e s Figure I I - 8 . R o t a t i n g reference frame model i l l u s t r a t i n g the s a t u r a t i o n recovery T i experiment. along the z - a x i s , no s i g n a l i s detected by the spectrometer, which i s designed to respond only to that component of the magnetization which l i e s i n the x,y-plane. Therefore, to assay the amount of magnetization along the z-axis at any p a r t i c u l a r time i t i s necessary to t i p the magnetization v e c t o r through 90° i n t o the x,y-plane. This i s accom-p l i s h e d by a p p l y i n g a s u i t a b l e amount of r a d i o frequency power at the appropriate frequency, i n the form of a short p u l s e , to t i p the magnet-i z a t i o n through 90° (a 90° p u l s e ) . In the r o t a t i n g reference frame, i n i t i a l l y the s p i n system i s at thermal e q u i l i b r i u m w i t h i t s l a t t i c e , and the magnetic moment i s d i r e c t e along the +z-axis. The e q u i l i b r i u m i s then destroyed by applying a 180 pulse of power which t i p s the magnetization i n t o the -z d i r e c t i o n . Spi l a t t i c e r e l a x a t i o n then r e s t o r e s the system to thermal e q u i l i b r i u m and 98 the amount of magnetization present at any p a r t i c u l a r time can be assayed by a p p l y i n g a 90° pulse to the r e s i d u a l component i n t o the x,y-plane. I f the magnetization along the z-axis i s sampled at s e v e r a l known delay times, t , a f t e r the i n i t i a l 180° p u l s e , then a p l o t of magnetization, Mo versus t , w i l l give the decay curve shown i n Figure I I - 9 . Each p o i n t on the curve must be obtained i n d i v i d u a l l y f o r progressive increments of t ; and a delay time of 5 or more Tj ( r e l a x a t i o n time = periods must be l e f t between each 180°-90° pulse sequence, to ensure that thermal e q u i l i b r i u m i s r e s t o r e d before the s t a r t of the next measurement. The r e l a x a t i o n r a t e i s most conveniently obtained by making a l i n e a r p l o t of " ^ ( M ^ - M )" vs " t " which gives the r e l a x a t i o n r a t e from the slope. The experiment, j u s t d e s c r i b e d , i s termed the " i n v e r s i o n recovery e x p e r i -ment" and although other r e l a x a t i o n experiments e x i s t t h i s was the one Figure I I - 9 . P l o t of magnetization versus t/T\ showing exponential recovery of magnetization. used to obt a i n the data i n s e c t i o n H C ( i i ) . I f one wants to estimate the r e l a x a t i o n r a t e without data process-i n g , the delay time t required to n u l l the resonance, that i s , the time when Mg = 0, can be used to o b t a i n the r e l a x a t i o n r a t e from the simple equation (4) This n u l l p o i n t method i s r a p i d and contrary to much p r e j u d i c e , i t 28 appears to provide r a t h e r accurate R^  v alues. 100 References 1. B. W. Rockett and G. Marr, J . Organomet. Chem. 167, 53(1979). 2. E. I. Edwards, R. Epton and G. Marr, i b i d . , 10_7 , 351(1976). 3. K. Tsubakihama, J . Matsua, T. Sasaki, K. Yoshida, T. Fujimura and K. A r a k i , J . Poly S c i . , JL7, 173(1979); A. G a l , M. Cais and D. H. Kohn, J . App. Poly. S c i . , 22, 3449(1978). 4. J . C. Johnson, Metallocene Technology, Noyes Data Corporation, Park Ridge, New Jersey, 1973. 5. H. Brunner, Agnew. Chem. I n t e r n a t . E d i t . , 10, 249(1971). 6. M. C a i s , S. Dani, Y. Eden, 0. G a n d o l f i , M. Horn, E. E. Isaacs, Y. Josephy, Y. Saar, E. S l o v i a and L. Snarsky, Nature, 270, 534(1977). 7. G. Marr and B. W. Rockett, J . Organomet. Chem., 58, 323(1973). 8. M. Wenzel, E. Nipper and W. Klose, J . Nucl. Med., 18, 367(1977). 9. M. S. Wrighton, M. C. P a l a z z o t t o , A. B. Bo c a r s l y , J . M. B o l t s , A. B. F i s h e r and L. Nadjo, J . Am. Chem. S o c , 100, 7264(1978); J . M. B o l t s , A. B. Bo c a r s l y , M. C. P a l a z z o t t o , E. G. Walton, N. S. Lewis and M. S. Wrighton, i b i d . , 101, 1378(1979); Chem. Eng. News, Mar. 19, 1979, p. 25; A. W. C. L i n , P. Yeh, A. M. Yacynych and T. Kuwana, J . E l e c t r o a n a l . Chem., 84_, 411(1977). 10. M. Adam and L. D. H a l l , J . Chem. Soc. Chem. Comm., 865 (1979); M. Adam and L. D. H a l l , Can. J . Chem., ( i n p r e s s ) . 11. P. L. Pauson and W. E. Watts, J . Chem. S o c , 2990(1963). 12. D. Horton, Methods i n Carbohydrate Chemistry, Academic P r e s s , New York, 2, 433 (1963). 13. M. Bergmann and L. Zervas, Chem. Ber., 64B, 975(1931). 14. D. Lednicer and C. R. Hauser, Organic Synthesis C o l l e c t i v e Volume V, 434(1973). 15. L. W. H a l l , J r . , D i s s e r t a t i o n Abst., 20, 78(1959). 16. J . K. Kochi and G. S. Hammond, J . Chem. S o c , 3443(1953). 17. D. Lednicer, T. A. Mashburn, J r . , and C. R. Hauser, Organic Syn-t h e s i s C o l l e c t i v e Volume V, 621(1973). 18. M. J . Adam and L. D. H a l l , Carbohydr. Res., 68, C17(1979). 101 19. D. Horton, The Amino Sugars, Jeanloz (Ed.), Academic P r e s s , New York, IA, 70(1969). 20. G. D. Broadhead, J . M. Osgerby and P. L. Pauson, J . Chem. Soc., 650(1958). 21. A. Thompson and M. L. Wolfram, Methods i n Carbohydrate Research, Academic Press, New York, 2^ , 215(1963). 22. D. N. Kursanov, N. Parnes, N. M. Loim, N. E. Kolobova, I. B. Z l o t i n a , P. V. P e t r o v s k i i and E. I. Fedin, J . Organomet. Chem., 4_4, C15 (1972). 23. N. M. Loim, D. N. Kursanov, Z. N. Parnes, N. N. Sul Dino and E. I. Fedin, J . Organomet. Chem., j>2, C33(1973). 24. M. J . Adam and L. D. H a l l , J . Organomet. Chem., ( i n p r e s s ) . 25. L. D. H a l l , Chem. Soc. Rev., 4_, 401(1975); L. D. H a l l , Chemistry i n Canada, 28_, 19(1976); J . K. M. Sanders, Annual Reports Chem. Soc. B, 75, 3(1979). 26. K. F. Wong, Ph.D. t h e s i s , 1979, and references t h e r e i n . 27. L. D. H a l l , K. F. Wong, W. E. H u l l and J . D. Stevens, J . Chem. Soc. Chem. Commun., i n press. 28. L. D. H a l l and L. Colebrook, Can. J . Chem., (submitted). CHAPTER I I I CYANURIC CHLORIDE; A GENERAL REAGENT FOR THE CHEMICAL MODIFICATION OF CARBOHYDRATES I I I A : I n t r o d u c t i o n As s t a t e d e a r l i e r , a more v e r s a t i l e approach to the chemical modi-f i c a t i o n of carbohydrates i s c l e a r l y needed i n order to be able to d e r i v a t i z e carbohydrates w i t h a broader range of organic and i n o r g a n i c substances, and al s o to extend the work presented thus f a r . A f u t u r e a p p l i c a t i o n of t h i s research, and one of the main reasons f o r the work i n t h i s t h e s i s , i s shown s c h e m a t i c a l l y i n the f o l l o w i n g i l l u s t r a t i o n . The anchor i n t h i s case may be a la r g e macromolecular support matr i x such as s i l i c a , alumina, a p o l y s a c c h a r i d e , or even a p r o t e i n . The probe could be any of a number of spectroscopic probes ("tags" or 102 103 " r e p o r t e r " groups); f o r example, a s p i n - l a b e l n i t r o x i d e used i n e s r , a f l u o r e s c e n t probe, a metal, or even an nmr probe such as a deuterium or a f l u o r i n e atom. The receptor molecule or "hapten" (to use an immuno-l o g i c a l term) can a l s o vary g r e a t l y and access to groups such as dyes, f a t t y a c i d s , carbohydrates, and p r o t e i n s would be d e s i r a b l e . The "shark," would represent t y p i c a l l y an enzyme w i t h a strong b i n d i n g a f f i n i t y f o r the s p e c i f i c receptor group, but could a l s o represent any l e s s s p e c i f i c i n t e r a c t i o n or a s s o c i a t i o n o c c u r r i n g between two molecules. The r a t i o n a l e underlying an experiment being that the probe molecule would r e l a y back to the experimenter inform a t i o n about such an a s s o c i a t i o n . The areas of study which might b e n e f i t from t h i s technology would be as v a r i e d as the r e q u i r e d chemistry and could i n c l u d e the f i e l d s o f : a f f i n i t y chromatography f o r the p u r i f i c a t i o n of enzymes and p r o t e i n s ; antibody-antigen i n t e r a c t i o n s t u d i e s ; substrate-enzyme b i n d i n g ; l i p i d -b i l a y e r membrane f u n c t i o n s ; and p o s s i b l y i n the study of c e l l - c e l l i n t e r a c t i o n s . I t may be advantageous to have the anchor support matr i x but t h i s i s not a mandatory p r e r e q u i s i t e f o r a l l of these i n v e s t i g a t i o n s . C l e a r l y m u l t i v a l e n t reagents are required f o r these s t u d i e s i n order to combine a l l of the d e s i r e d e n t i t i e s . In the present work, the chemistry of one such m u l t i v a l e n t reagent, cyanuric c h l o r i d e , i s i n v e s t i -gated w i t h p a r t i c u l a r a p p l i c a t i o n s to carbohydrate chemistry. Cyanuric c h l o r i d e [1] i s a h e t e r o c y c l i c compound c o n t a i n i n g three c h l o r i n e s u b s t i t u e n t s each of which can be d i s p l a c e d by any of a v a r i e t y of n u c l e o p h i l e s under a wide range of c o n d i t i o n s . This reagent has been known s i n c e 1827"^ and i s now very f u l l y documented. Owing to i t s low cost and v e r s a t i l i t y , t h i s reagent has found uses i n an extremely wide range of areas: "medicine," " i n d u s t r y , " "chemistry," and "biochemistry." 104 In "medicine," cyanuric c h l o r i d e has found uses i n the development 1 2 of b a c t e r i o c i d e s and anti- c a n c e r drugs ' . Quinoline d e r i v a t i v e s [ 2 ] , a r s e n i c and antimony compounds [3] and [ 4 ] , and t r i e t h y l e n e melamine [5] are t y p i c a l examples; compounds [3] and [5] have been shown to have d e s i r a b l e chemotherapeutic p r o p e r t i e s . Compound [5] has been subject 2 to a considerable study and was shown to i n h i b i t growth of c e r t a i n types of tumors and to be of b e n e f i t i n treatment of leukemia; ( i t i s a l s o known to cause cancer!). In " i n d u s t r y " 1 , substances c o n t a i n i n g the s - t r i a z i n e s t r u c t u r e have been used as p e s t i c i d e s and p r e s e r v a t i v e s . Compounds of the aryloxyamino s u l f o n i c a c i d type condensed w i t h cyanuric c h l o r i d e are u s e f u l as moth-proof ers and f i b r o u s p r o t e c t o r s . In the rubber i n d u s t r y , c e r t a i n t r i a z i n e d e r i v a t i v e s are u s e f u l as a n t i o x i d a n t s and v u l c a n i z a t i o n a c c e l e r -a t o r s . Resins and p l a s t i c s have al s o been formed from cyanuric c h l o r i d e , 3 the most w e l l known of which are the melamine-formaldehyde polymers [6] ; (these form hard p l a s t i c s and are used i n making dinnerware and decorative surface coatings f o r counter tops, t a b l e s , and w a l l coverings under the brand name of Formica). The melamine necessary f o r t h i s p l a s t i c i s formed by the r e a c t i o n of cyanuric c h l o r i d e w i t h ammonia. Cyanuric c h l o r i d e has a l s o been used i n the manufacturing of ex p l o s i v e s (such as 105 cyanuric t r i a z i d e ) and i n the formation of surface a c t i v e agents by the r e a c t i o n of cyanuric c h l o r i d e w i t h dodecylaminoethylsulfate (C 1 2H25NHCH2CH20-S0 3Na). P o s s i b l y though, the s i n g l e most important i n d u s t r i a l a p p l i c a t i o n to date, and a l s o the most important w i t h respect to t h i s chapter, i s 4 the use of ^ - t r i a z i n e dyes as r e a c t i v e dyes f o r c e l l u l o s i c f i b e r s . Replacement of one or two c h l o r i n e s u b s t i t u e n t s by the amino group of an aromatic dye gives m a t e r i a l s such as the P r o c i o n dyes [7] and [ 8 ] ; these were f i r s t introduced by ICI i n 1956. I t i s i n t e r e s t i n g to note that the s _ - t r i a z i n y l r i n g e f f e c t i v e l y acts as a "chromophoric block" and e l e c t r o n i c a l l y " i n s u l a t e s " the two chromophoric groups. Therefore i f 106 a blue dye i s put on one p o s i t i o n and a yel l o w dye on a second p o s i t i o n of the j B - t r i a z i n y l moiety, the r e s u l t a n t r e a c t i v e dye i s green i n c o l o r . Because they have s u p e r i o r wet fastness the increased use of these dyes has caused a cu r t a i l m e n t i n the use of other non c o v a l e n t l y bonded dyes and i n some cases replaced them a l t o g e t h e r . I n t e r e s t i n g l y , one of these dyes (Cibacron Blue) [9] has r e c e n t l y been shown to be a u s e f u l reagent i n a f f i n i t y chromatography"'; when coupled to Sepharose beads (a cross l i n k e d polysaccharide used i n CI 107 chromatography) i t i s u s e f u l f o r the p u r i f i c a t i o n of c e r t a i n enzymes. This i s another encouraging r e s u l t as i t r e l a t e s w e l l to the present chapter and i t i s an area to which t h i s t h e s i s work was hoped to advance. Other chromatographic a p p l i c a t i o n s i n v o l v i n g cyanuric c h l o r i d e have r e c e n t l y appeared. For the most part these have i n v o l v e d the immobil-i z a t i o n of enzymes such as t r y p s i n and e s t e r a s e ^ f o r use i n a f f i n i t y chromatography. Other i n t e r e s t i n g recent work us i n g cyanuric c h l o r i d e 7 8 i n c l u d e s the b i n d i n g of: NAD to dextran ; gl u t a t h i o n e to c e l l u l o s e ; 9 f l u o r e s c e n t t r i a z i n e reagents to p r o t e i n s ; polyethylene g l y c o l to p r o t e i n s 1 ^ ; a n t i m i c r o b i a l compounds to c e l l u l o s i c f i b e r s ; the d e r i v -a t i z a t i o n of e l e c t r o d e surfaces w i t h ferrocene"'""'" has already been men-tio n e d i n chapter (HA) and w i l l be discussed again l a t e r i n t h i s one. Of the very recent work, the most s i g n i f i c a n t , as i t a p p l i e s to t h i s chapter, was reported by Bishop et a l . where p o l y s a c c h a r i d e - p r o t e i n 12 13 conjugates and monosaccharide-protein conjugates , f o r immunological studies,were formed using cyanuric c h l o r i d e . From a h i s t o r i c a l stand-point i t i s noteworthy that the l a t t e r represents the f i r s t reported synt h e s i s of a monosaccharide (2-amino-2-deoxy-D-glucopyranose)-s-triazine compound. In l i g h t of the s t u d i e s mentioned above i t w i l l be c l e a r why t r i a -z i n e reagents were chosen as being worthy of f u r t h e r study, and i t was the i n t e n t i o n of the present study to develop the use of cyanuric c h l o r i d e to form novel conjugates of carbohydrates w i t h i n t e r e s t i n g p r o p e r t i e s and uses. The sequence of the f o l l o w i n g d i s c u s s i o n i s as f o l l o w s . F i r s t , the r e a c t i v i t y of cyanuric c h l o r i d e toward n u c l e o p h i l i c displacement w i l l be discussed i n the context of four groups of 108 compounds; (a) monosaccharides, (b) hydrophobic a l k y l molecules, (c) metal compounds, and (d) s p i n l a b e l n i t r o x i d e s . In each case the r e a c t i o n c o n d i t i o n s and order of a d d i t i o n w i l l be examined. The syn-theses i n t h i s s e c t i o n w i l l be culminated w i t h an example of the forma-t i o n of s y n t h e t i c " g l y c o l i p i d s . " Next, the chemical m o d i f i c a t i o n of polysaccharides w i l l be discussed and emphasis w i l l be given w i t h regard to s p i n l a b e l l i n g of these m a t e r i a l s ; a t t e n t i o n w i l l a l s o be given as to how s p i n l a b e l l i n g can be used i n s t r u c t u r e - d e t e r m i n a t i o n of these m a t e r i a l s and a l s o as a model " t e s t v e h i c l e " f o r i n t r o d u c i n g other sub-stances i n t o polysaccharides v i a cyanuric c h l o r i d e . Information as to whether the l a b e l i s bound or unbound, the q u a n t i t y attached, the d i s -tance between two n i t r o x i d e s and the m o b i l i t y of the l a b e l s w i l l be discus s e d . The two parent s p i n l a b e l n i t r o x i d e molecules used i n t h i s chapter are shown below. These are h e t e r o c y c l i c p y r r o l i d i n e - l - o x y l d e r i v a t i v e s ( = H 2 N S I ) I ( = H 0 S I ) [10] [11] which c o n t a i n an unpaired e l e c t r o n making them paramagnetic and there-f o r e d e t e c t a b l e by esr spectroscopy. Such n i t r o x i d e s are amongst the most s t a b l e of f r e e r a d i c a l s and many chemical m o d i f i c a t i o n s of the f u n c t i o n a l groups at p o s i t i o n A can be e f f e c t e d without d e s t r u c t i o n of the paramagnetic center. The ab b r e v i a t i o n s f o r these l a b e l s w i l l be 109 used throughout t h i s chapter. For background i n f o r m a t i o n the reader i s r e f e r r e d to s e c t i o n ( H I E ) which describes the esr spectroscopy of n i t r o x i d e s . The s p i n l a b e l l i n g of Bovine Serum Albumin (BSA), aluminum oxide, and glass w i l l a l s o be demonstrated to i l l u s t r a t e t hat t r i a z i n e -n i t r o x i d e chemistry i s a l s o u s e f u l o u t side the realm of carbohydrate chemistry. F i n a l l y , the two spectroscopic techniques, nmr and e s r , w i l l be discussed as they provide the necessary s t r u c t u r a l i n f o r m a t i o n f o r the a n a l y s i s of these compounds. F i r s t , the use of nmr spectroscopy w i l l be b r i e f l y described as a s t r u c t u r a l t o o l f o r both the diamagnetic and paramagnetic monomeric compounds synthesized i n ( I I I B ) . Then a more d e t a i l e d d i s c u s s i o n of esr spectroscopy of n i t r o x i d e s w i l l be presented; t h i s w i l l i n c l u d e some of the necessary theory and r e s u l t s needed f o r the p r a c t i c i n g chemist (as d i s t i n c t from the esr s p e c t r o s c o p i s t ) to understand the r e s u l t s presented p r e v i o u s l y . I I I B : Synthesis ( i ) Nitrogen Nucleophiles I t i s known that replacement of one c h l o r i n e s u b s t i t u e n t i n cyanuric c h l o r i d e by a primary or secondary amine, a hydrazine, or a r e l a t e d compound, d e a c t i v a t e s the remaining c h l o r i n e s u b s t i t u e n t s ; as a r e s u l t stepwise displacement r e a c t i o n s can be achieved. These d i s -placement r e a c t i o n s are f o r m a l l y analogous to n u c l e o p h i l i c aromatic 14 s u b s t i t u t i o n as shown below . The n i t r o g e n atoms i n the h e t e r o c y c l i c r i n g act as e l e c t r o n withdrawing groups, i n much the same f a s h i o n as the n i t r o s u b s t i t u e n t s on a benzene r i n g , thereby s t a b i l i z i n g the "carbanion" t r a n s i t i o n s t a t e and thus g i v i n g the c h l o r i n e s u b s t i t u e n t s of cyanuric c h l o r i d e t h e i r i n i t i a l l y high r e a c t i v i t y to n u c l e o p h i l i c 110 G withdraws electrons: stabilizes and activates G donates electrons: destabilizes and deactivates displacement ( r e a d i l y hydrolyzed by H 20). S u b s t i t u t i o n of a c h l o r i n e atom by an e l e c t r o n donating group such as an amine, d e s t a b i l i z e s the t r a n s i t i o n s t a t e and thereby d e a c t i v a t e s the remaining c h l o r i n e s u b s t i t -uents. In c o n t r a s t , oxygen and s u l f u r s u b s t i t u e n t s have a very weak d e a c t i v a t i n g e f f e c t and t h e i r r e a c t i o n s w i l l be discussed s e p a r a t e l y l a t e r . The e m p i r i c a l " r u l e of thumb," u s u a l l y expressed i n the l i t e r a t u r e , to the stepwise s u b s t i t u t i o n of the c h l o r i n e s u b s t i t u e n t by amines i n aqueous media, i s shown i n the diagram below. This " r u l e , " that the I l l f i r s t c h l o r i n e atom i s replaced at 0°C, the second at 30-50°C, and the t h i r d at 90-100°C cannot be used g e n e r a l l y . F i r s t , i t a p p l i e s only to aqueous r e a c t i o n s , and other f a c t o r s such as b a s i c i t y of the amine, the s t e r i c bulk both of the s u b s t i t u e n t s already attached to the t r i a z i n e moiety and of the approaching amine, the d e a c t i v a t i n g e f f e c t of any amine s u b s t i t u e n t already attached, and the base acceptor used i n the r e a c t i o n medium, a l l play key r o l e s i n determining r e a c t i v i t y . Thus, t h i s over-s i m p l i f i e d " r u l e of thumb" i s at best an inaccurate summary. Some r e a c t i o n s , however, do f o l l o w t h i s scheme w e l l ; f o r example, the r e a c t i o n of cyanuric c h l o r i d e , f i r s t w i t h 4-amino 2 , 2 , 6 , 6 - t e t r a m e t h y l p i p e r i d i n e - l -o x y l [10] at 0°C i n aqueous acetone w i t h sodium bicarbonate to form com-pound [12], and then subsequent r e a c t i o n s w i t h e i t h e r n-hexylamine or dodecylamine i n the same solvent and base acceptor at 45°C and 80°C to form compounds [13], [14], and [15]. Compound [11] had been p r e v i o u s l y made by L i k h t e n s h t e i n i n 1969"^, but the present p r e p a r a t i o n (see E x p e r i -mental) i s much simp l e r . A l l of the s p i n - l a b e l l e d products were pink or orange i n colour due to the presence of the n i t r o x i d e moiety; they could be p r e c i p i t a t e d d i r e c t l y from the r e a c t i o n mixture and r e q u i r e d no f u r t h e r p u r i f i c a t i o n except to be dried-under vacuum at V50°C. An i n t e r e s t i n g point should be made here, concerning the order of a d d i t i o n of s u b s t i t u e n t s to the ^ - t r i a z i n e center. In the above-mentioned sequence of r e a c t i o n s i t was found that the s p i n l a b e l n i t r o x i d e [10] must be attached to the s - t r i a z i n e r i n g i n the f i r s t step. Attempts to react the amino s p i n l a b e l [10] w i t h e i t h e r the mono- or the d i - a l k y l c h l o r o - t r i a z i n e d e r i v a t i v e s f a i l e d . Other examples i l l u s t r a t i n g the importance of the order of a d d i t i o n w i l l be discussed as they appear. S u b s t i t u t i o n of s u i t a b l y f u n c t i o n a l i z e d aromatic TT-bonded NHSI M O T R N H , 4 5 ° C ^^^N R= n-Hexyl [13] [12] u , n / f l f . M a n r n . if - n-Dodecyl [14] H 20/Ace , NaHC03 ' N R H NHSI [14] CHzsNHj 80°C N H,0/Ace, NaHCO, , . ^ ,, r f e Q z N ^ N x N - C l 2 H 2 5 [15] 113 organometallic complexes are a l s o f a c i l e as shown i n the f o l l o w i n g r e a c t i o n of p - t o l u i d i n e chromium t r i c a r b o n y l [16] w i t h cyanuric c h l o r i d e to produce [17]. This r e a c t i o n proceeds as smoothly as the A-amino s p i n [1] Me /CN„ CO CO CO [16] H2OA NaHCO, ce CI N' [17] CO CO CO l a b e l r e a c t i o n and although no subsequent r e a c t i o n s were performed on [17], i t s d e r i v a t i z a t i o n i n any of the ways shown i n t h i s chapter should pose no problems. Ferrocene can a l s o be attached to cyanuric c h l o r i d e and t h i s r e a c t i o n i s discussed i n s e c t i o n I I I C . Reactions of amino sugars with cyanuric c h l o r i d e are a l s o q u i t e f a c i l e and t y p i c a l examples are shown i n the f o l l o w i n g r e a c t i o n schemes. 114 13 As s t a t e d before, compound [20] was f i r s t prepared by Bishop and the l i t e r a t u r e s y n t h e s i s was repeated here s u c c e s s f u l l y . Reaction of the blocked amino sugar [19] was performed i n the same way wi t h good r e s u l t s and a y i e l d of 76%. A l l attempts to react [21] w i t h the 4-amino n i t r o x -ide [10] or to react the s p i n l a b e l l e d t r i a z i n e d e r i v a t i v e [12] with the blocked amino sugar [19] however, f a i l e d . This seems to r e f l e c t the low HNGIC . X . [2i: NHSI N ^ ^ N , f ^ 1 V r NO Reaction CI [10] XX + CI ^ C l N < ^ N , / \ j U M e \ / ^ NO Reaction AcO N H 3 B r [12] [19] b a s i c i t y of both the amino sugar and the s p i n l a b e l ; however, t h i s same amino sugar re a c t s r e a d i l y w i t h the oxygen l i n k e d s p i n l a b e l d e r i v a t i v e [22] to form [23] as shown below. 115 This oxygen l i n k e d s p i n l a b e l l e d t r i a z i n e d e r i v a t i v e [22] a l s o r e a c t s more r e a d i l y under milder c o n d i t i o n s w i t h n-hexylamine than does i t s n i t r o g e n l i n k e d counterpart [12] as shown below: OSI OSI ^ N X C H N H H 2 0/Ace 0°C N ^ N I I + NaHC0..20min * J f II [22] [24] NHSI NHSI N ^ N , r u K l u H 2 0 /Ace 20m in N ^ k N 1 1 + C ^ H « N H 2 45OCN3HC03 * JL II C I ^ ^ N ^ ^ C I cK ^ N C 6 H I 3 H [12] [13] These examples serve both to i l l u s t r a t e that the d i f f e r e n c e i n r e a c t i v i t y between oxygen and n i t r o g e n s u b s t i t u t e d c h l o r o t r i a z i n e s i s s u b s t a n t i a l and al s o to ca r r y the reader i n t o the next s e c t i o n , i n which the r e a c t i o n s of hydroxyl and t h i o l n u c l e o p h i l e s w i t h cyanuric c h l o r i d e w i l l be d i s -cussed more thoroughly. ( i i ) Oxygen and S u l f u r Nucleophiles As mentioned p r e v i o u s l y , oxygen and s u l f u r s u b s t i t u e n t s have a much smaller d e a c t i v a t i n g e f f e c t on the r e a c t i v i t y of remaining c h l o r i n e atoms of s u b s t i t u t e d t r i a z i n e u n i t s than do t h e i r n i t r o g e n counterparts. Thus one can, as i l l u s t r a t e d i n the example above, react an amine with an oxygen l i n k e d c h l o r o t r i a z i n e d e r i v a t i v e under c o n d i t i o n s s i m i l a r to those that would be used w i t h cyanuric c h l o r i d e i t s e l f , and not the ^ 50°C needed i f s u b s t i t u t i o n by a ni t r o g e n moiety preceeds i t . This increased l e v e l of r e a c t i v i t y has i t s obvious advantages; however, i t a l s o has disadvan-116 tages. I f t h i s chemistry i s to be used to d e r i v a t i z e p r o t e i n s or other s e n s i t i v e b i o l o g i c a l molecules the r e a c t i o n c o n d i t i o n s must be kept m i l d and temperatures above 50°C w i l l f r e q u e n t l y be u n d e s i r a b l e . Should the t r i a z i n e nucleus be already d i s u b s t i t u t e d with two N-linked groups i t i s l i k e l y that the subsequent coupling r e a c t i o n could r e q u i r e ^100°C; however, by having one or two of these s u b s t i t u e n t s l i n k e d v i a oxygen or s u l f u r , the temperature of the subsequent coupling r e a c t i o n may be kept below 50°C. Even i n non s e n s i t i v e systems where there are no tempera-ture l i m i t a t i o n s t r i s u b s t i t u t i o n w i t h an amine or d i a m i n o c h l o r o t r i a z i n e i s sometimes impossible f o r one reason or another and t h e r e f o r e s u i t a b l e oxygen or s u l f u r s u b s t i t u e n t s may provide a v i a b l e procedure. The main disadvantage i n t h i s chemistry however, i s that because there i s very l i t t l e d e a c t i v a t i o n of the _ s - t r i a z i n e r i n g by an oxygen or s u l f u r sub-s t i t u e n t , r e a c t i o n s of these n u c l e o p h i l e s w i t h cyanuric c h l o r i d e or d i c h -l o r o t r i a z i n e compounds can sometimes lead to o v e r - s u b s t i t u t i o n and hence to mixtures of compounds from which p u r i f i c a t i o n of a s i n g l e compound can be d i f f i c u l t . This problem i s e x e m p l i f i e d by the r e a c t i o n of cyanuric c h l o r i d e w i t h the A-hydroxy s p i n l a b e l [11]. While r e a c t i o n of cyanuric 117 c h l o r i d e w i t h one equivalent of the 4-hydroxy n i t r o x i d e [11] y i e l d s [22] and two equiv a l e n t s y i e l d s [25], r e a c t i o n c o n d i t i o n s must be c a r e f u l l y c o n t r o l l e d to prevent mixtures of products from being formed. Both r e a c t i o n s are c a r r i e d out by the slow dropwise a d d i t i o n (40 min to 1 h) of the 4-hydroxy s p i n l a b e l i n sodium hydroxide s o l u t i o n to a s t i r r e d s o l u t i o n of cyanuric c h l o r i d e i n i c e - c o l d acetone. A d d i t i o n must be made slo w l y i n order to keep the concentration of spin l a b e l low ( i t reacts almost immediately) and thereby to suppress the unwanted formation of the b i r a d i c a l ( i n the case of the monoradical preparation) or the t r i r a d i c a l i n the case of the b i r a d i c a l p r e p a r a t i o n . The y i e l d s f o r both these r e a c t i o n s are low (10-16%) probably because both cyanuric c h l o r i d e and the products are hydrolyzed r e a d i l y by sodium hydroxide even at 0°C; ne v e r t h e l e s s , the products can be f i l t e r e d d i r e c t l y from the r e a c t i o n mixture, and only r e q u i r e d r y i n g at ^ 50°C (to sublime away any unreacted cyanuric c h l o r i d e ) f o r p u r i f i c a t i o n . This ease of product i s o l a t i o n and the low cost of the s t a r t i n g m a t e r i a l s help to make these low y i e l d s t o l e r a b l e . The r e a c t i o n of hydroxymethyl ferrocene [26] (see IIB) w i t h cyanuric c h l o r i d e discussed e a r l i e r i n the t h e s i s , i s s i m i l a r to that of the 4-hydroxy s p i n l a b e l . In t h i s r e a c t i o n a s o l u t i o n of the a l c o h o l [26] i n [1] + CH 2 0H H 20/Ac OFc NaOH, 0°C JL CI N CI (==FcCH2OH ) [26] cr N [27] 118 acetone and an aqueous s o l u t i o n of sodium hydroxide were added separately over a period of 1 h; a f t e r f i l t r a t i o n of a by-product (see IIB) the f i l t r a t e was allowed to become a c i d i c upon standing (because of h y d r o l -y s i s of c yanuric c h l o r i d e ) and the r e s u l t i n g yellow p r e c i p i t a t e f i l t e r e d . No d i s u b s t i t u t e d product was detected. In the above, r e a c t i o n and syn- . t h e s i s of the d i s u b s t i t u t e d b i s - f e r r o c e n e d e r i v a t i v e was not attempted. Because of a recent i n t e r e s t i n c o a t i n g e l e c t r o d e surfaces w i t h f e r r o -cene1"'", t h i s product may f i n d an immediate a p p l i c a t i o n . Reaction of the f r e e hydroxyl group of a s u i t a b l y blocked mono-saccharide w i t h a c h l o r o t r i a z i n e d e r i v a t i v e i s e x e m p l i f i e d by the f o l -lowing r e a c t i o n : Reaction of 1,2:3,4-di-O-isopropylidene-a-g-galcactopyranose [28] with the oxygen l i n k e d s p i n l a b e l l e d s - t r i a z i n e [22] was c a r r i e d out conven-i e n t l y i n reagent grade benzene with, powdered sodium hydroxide, to y i e l d 119 compound [29]. When sodium hydroxide was replaced by sodium carbonate there was no detectable r e a c t i o n . Compound [29] was not i s o l a t e d but i n s t e a d was reacted d i r e c t l y w i t h n-hexylamine i n aqueous acetone to give the t r i s u b s t i t u t e d d e r i v a t i v e [30] i n 45% o v e r a l l y i e l d , without r e a c t i o n temperatures ever exceeding room temperature! This r e a c t i o n i l l u s t r a t e s the u t i l i t y of the s - t r i a z i n e moiety to serve as a " t r i -v a l e n t l o c u s " ; being used here to form a s y n t h e t i c g l y c o l i p i d complete w i t h spectroscopic "tag". In r e a l i t y t h i s p a r t i c u l a r molecule would not be very u s e f u l as i t stands, since l i p i d s g e n e r a l l y have longer a l k y l chains ( t y p i c a l l y C16) and the sugar moieties of g l y c o l i p i d s contain unblocked " f r e e " hydroxyl groups. Meeting these requirements w i t h t r i a z i n e chemistry i s not viewed as a problem and one such more r e a l -i s t i c s y n t h e t i c g l y c o l i p i d w i l l be discussed a f t e r the r e a c t i o n s of s u l f u r n u c l e o p h i l e s and cyanuric c h l o r i d e have been presented. Reaction of the blocked galactose [28] d i r e c t l y w i t h cyanuric c h l o r i d e was a l s o attempted but i s o l a t i o n of the product was not achieved owing to i t s high r e a c t i v i t y (the product decomposed on s i l i c a g e l ) . T h i s , however, does not prevent the subsequent d e r i v a t i z a t i o n of t h i s crude product as was done i n the case of compound [29]. T h i o l s a l s o react w e l l w i t h cyanuric c h l o r i d e and t h i s i s exempli-f i e d by v a r i o u s r e a c t i o n s of 2 , 3 , 4 , 6 - t e t r a - 0 - a c e t y l - l - t h i o - 8 - D - g l u c o -pyranose [31] w i t h c h l o r o t r i a z i n e s . The -SH group i s an extremely good n u c l e o p h i l e and has an advantage over i t s -OH counterpart i n that i t does not r e q u i r e a strong base to f i r s t remove the attached proton. However, the weak d e a c t i v a t i n g e f f e c t of the s u l f u r s u b s t i t u e n t on the s - t r i a z i n e r i n g was not d i s c e r n i b l y d i f f e r e n t from the a l c o h o l s u b s t i t u e n t e f f e c t . Three equivalents of the 1-thio-glucose [31] was reacted with 120 cyanuric c h l o r i d e i n a c e t o n i t r i l e and t r i e t h y l amine to form the sym-m e t r i c a l t r i - s a c c h a r i d e d e r i v a t i v e [32]. The r e a c t i o n was immediate SGIc , , / ^ ° V S H CH 3CN r ^ OAc H I [31]l=HSGlc) 1321 and, a f t e r pouring i n t o i c e water, the p r e c i p i t a t e was f i l t e r e d and d r i e d to y i e l d the pure product. Reaction of one equivalent of the 1-thio-sugar [31] to form the monosubstituted d i c h l o r o compound was unsuccessful owing to the high r e a c t i v i t y of that product and i t s decomposition s i m i l a r to that of the mono-galactose p r e p a r a t i o n . The 1-thio-glucose [31] was a l s o reacted w i t h the n i t r o g e n and oxygen l i n k e d s p i n l a b e l l e d s_-triazine compounds [12] and [22] to form the mono- and b i s - t h i o sugar d e r i v a t i v e s of each. Due to the good n u c l e o p h i l i c i t y of the t h i o sugar these reac-t i o n s a l l went r e a d i l y at room temperature, even w i t h the amino s p i n l a b e l d e r i v a t i v e [12]. Mixtures of mono- and b i s - s u b s t i t u t i o n by the t h i o sugar d i d not seem to pose any problems (not d e t e c t a b l e on t i c ) and the mono-substituted t h i o glucose products [33] and [34] were i s o l a t e d e a s i l y ; t h i s apparent d e a c t i v a t i o n by the f i r s t s u l f u r s u b s t i t u e n t may be caused more by s t e r i c f a c t o r s than anything e l s e . The ferrocene t r i a z i n e d e r i v a t i v e [27] a l s o r e a c t s r e a d i l y w i t h t h i o - g l u c o s e to form the metal sugar conjugate [37]. More inf o r m a t i o n OFc p O A c N ^ N , / ° \ ? H C H 3 C N OFc OAc 1271 [ 3 1 ] [ 3 7 ] about t h i s d e r i v a t i v e has been given i n ( I I B ) . As mentioned e a r l i e r , t h i s p a r t i c u l a r chemistry i n v o l v i n g t h i o glucose, was extended to form s y n t h e t i c g l y c o l i p i d s . In a p r e l i m i n a r y study, a f o u r t h year undergraduate student, A r t van der E s t , while work-ing i n Dr. H a l l ' s l a b o r a t o r y , p r e p a r e d 1 ^ one such s y n t h e t i c g l y c o l i p i d from the f o l l o w i n g sequence of r e a c t i o n s . Although elemental a n a l y s i s was not obtained f o r the f i n a l product, the intermediates up to [33] gave acceptable elemental a n a l y s i s and compounds [38] and [39] appeared to be homogeneous by t i c ; furthermore, esr i n t e g r a t i o n gave a q u a n t i t a t i o n of sp i n l a b e l to w i t h i n 10% of the c a l c u l a t e d value. This f i n a l product was 122 then incorporated i n t o liposome model membranes, ( i i i ) Summary Cyanuric c h l o r i d e can be r e a d i l y s u b s t i t u t e d by the amino, hydroxy and t h i o groups of many compounds and under a v a r i e t y of r e a c t i o n condi-t i o n s . Both aqueous and non-aqueous media can be used along with a number of base acceptors i n c l u d i n g : sodium bicarbonate, sodium carbonate, sodium hydroxide, and t r i e t h y l a m i n e . Amines add to the t r i a z i n e r i n g i n a pronounced stepwise a d d i t i o n . Each a d d i t i o n d e a c t i v a t e s the r i n g a l l o w i n g i t to be s u b s t i t u t e d by three 123 d i f f e r e n t amino groups. Alkoxy and t h i o s u b s t i t u e n t s , however, have a much l e s s e r d e a c t i v a t i n g e f f e c t and both advantages and disadvantages accrue from t h e i r use. Due to t h e i r low d e a c t i v a t i n g e f f e c t , r e a c t i o n s between cyanuric c h l o r i d e and hydroxyl and thio-compounds can r e s u l t i n mixtures; however, i t was found that t h i s d i d not occur i n most cases and i t could be c o n t r o l l e d by choosing the appropriate r e a c t i o n condi-t i o n s . An obvious advantage of an alkoxy or t h i o s u b s t i t u t e d c h l o r o -t r i a z i n e compound i s i n i t s much higher r e a c t i v i t y i n subsequent nucleo-p h i l i c displacement r e a c t i o n s as compared to that of i t s amino l i n k e d analogue. The order of s u b s t i t u t i o n onto the t r i a z i n e r i n g i s als o q u i t e important. Since many f a c t o r s c o n t r i b u t e to the success or f a i l u r e of these s u b s t i t u t i o n s , i t i s d i f f i c u l t , i f not imp o s s i b l e , to p r e d i c t beforehand the c o r r e c t sequence of a d d i t i o n to prepare d i - or t r i -s u b s t i t u t e d t r i a z i n e s . A few general observations can, however, be made: weakly b a s i c amines, such as the s p i n l a b e l [10] and amino-sugars do not react w e l l with amine s u b s t i t u t e d c h l o r o t r i a z i n e s and should e i t h e r be added f i r s t , or a f t e r an alkoxy or t h i o s u b s t i t u t i o n ; when using s t r o n g l y a l k a l i n e c o n d i t i o n s to f a c i l i t a t e s u b s t i t u t i o n w i t h hydroxyl c o n t a i n i n g compounds, s u b s t i t u t i o n a l s o should e i t h e r be made f i r s t , or f o l l o w i n g an alkoxy or t h i o s u b s t i t u t i o n , since elevated tem-peratures may cause competing h y d r o l y s i s ; r e l a t i v e l y b a s i c amines, such as alkylamines, and a l s o t h i o d e r i v a t i v e s , can u s u a l l y s u b s t i t u t e the r i n g at any stage regardless of the s u b s t i t u e n t s p r e v i o u s l y attached. In t h i s study i t has been shown that monosaccharides can be l i n k e d to the t r i a z i n e r i n g through n i t r o g e n , oxygen and s u l f u r atoms. As w e l l as monosaccharides, a host of i n t e r e s t i n g groups i n c l u d i n g a l k y l , s p i n 124 l a b e l and organo-metallic moieties have a l s o been attached to the r i n g i n v a r i o u s combinations. An example of how such a combination might y i e l d u s e f u l compounds was shown by the production of " s y n t h e t i c g l y c o l i p i d s . " Other p o s s i b l e a p p l i c a t i o n s are numerous and i n the next s e c t i o n i t w i l l be shown how t h i s chemistry can be a p p l i e d to chemically modify poly-saccharides and other macromolecules and s u r f a c e s . One p a r t i c u l a r l y c h a l l e n g i n g extension of t h i s work that the author u n f o r t u n a t e l y was unable to pursue due to time l i m i t a t i o n s , i s the p o s s i b l e formation of c h l o r o t r i a z i n e - m e t a l chelate d e r i v a t i v e s . Some p o s s i b l e s t r u c t u r e s are shown s c h e m a t i c a l l y i n the f o l l o w i n g i l l u s -t r a t i o n . Just as s p i n l a b e l s can be used as "probes," so a l s o can metal d e r i v a t i v e s . Such reagents may be u s e f u l as: metal probes f o r b i o l o g -i c a l systems; heavy metal c a r r i e r s f o r e l e c t r o n microscopy s t u d i e s ; pharmaceuticals when l i n k e d to s u i t a b l e molecules such as sugars; and even used i n a f f i n i t y chromatography, when l i n k e d to supports such as p o l y s a c c h a r i d e s , f o r the p u r i f i c a t i o n of p r o t e i n s or other metal b i n d i n g substances. There i s considerable i n t e r e s t i n the use of metal compound f o r these purposes and f u r t h e r work using cyanuric c h l o r i d e may be f r u i t f 125 I I I C : Macromolecule and Surface M o d i f i c a t i o n ( i ) Polysaccharides (a) The General Reaction In t h i s s e c t i o n i t w i l l be demonstrated how cyanuric c h l o r i d e can be used to chemically modify polysaccharides i n a completely general way. The a c t u a l r e s u l t s presented w i l l be r e s t r i c t e d to s p i n l a b e l l i n g v i a cyanuric c h l o r i d e ; however, the use of s p i n l a b e l l i n g as a t e s t v e h i c l e or "probe" f o r the general chemistry of cyanuric c h l o r i d e mediated d e r i v a t i z a t i o n of polysaccharides w i l l be shown. E s r - r e s u l t s and - s p e c t r a w i l l be shown and discussed where needed; l i t t l e e x planation i s given to the reader not f a m i l i a r w i t h n i t r o x i d e e s r , who i s i n s t e a d r e -f e r r e d to s e c t i o n H I E f o r a more d e t a i l e d explanation of that p a r t i c u -l a r aspect of n i t r o x i d e - e s r spectroscopy. The f a c t that cyanuric c h l o r i d e had p r e v i o u s l y been used success-f u l l y i n the d y e s t u f f s i n d u s t r y to form r e a c t i v e dyes f o r c e l l u l o s i c f i b e r s was very encouraging and provided a great deal of u s e f u l informa-t i o n concerning the r e a c t i o n and p r o p e r t i e s of cyanuric c h l o r i d e - d e r i v -a t i z e d c e l l u l o s e . The present work, as mentioned before, has been s o l e l y concerned w i t h the s p i n l a b e l l i n g of polysaccharides v i a cyanuric c h l o r i d e . The c h l o r o t r i a z i n e s p i n l a b e l s used i n t h i s study were discussed i n s e c t i o n s I I I B ( i ) and ( i i ) and are summarized below. The underlined a b r e v i a t i o n s f o r these compounds w i l l be used throughout t h i s chapter. A l l three l a b e l s [12], [22] and [25] were used to l a b e l v a r i o u s polysaccharides; however, more emphasis was placed on the use of the mono-radicals [12] and [22] s i n c e the esr spectra of b i r a d i c a l s are more complicated and l e s s f u l l y u n d e r s t o o d 1 7 . The t r i a z i n e n i t r o x i d e [12] was f i r s t prepared 126 NHSI N ^ N C K N [12] IHC1 2TNHSI) C K N ^ C l n ] [22] [5CI2OSU OS! C r N ^ O S l [26] IsClTOSl) by other workers as state d before i n I I I B and has been used to l a b e l 18 cotton f i b e r s i n order to study t h e i r s t r u c t u r e and conformation Although much l i t e r a t u r e has been published on the s p i n l a b e l l i n g of 19 p r o t e i n s and l i p i d s , there has been a general paucity of methodology s u i t a b l e f o r l a b e l l i n g polysaccharides u n t i l the f i r s t systematic study 20 was reported by A p l i n and H a l l The polysaccharides that have been s p i n l a b e l l e d using the t r i a z i n e chemistry were s e l e c t e d from a v a r i e t y of p l a n t , animal and b a c t e r i a l sources and t h e i r s t r u c t u r e s are shown i n Figure I I I - l . An abundance of hydroxyl f u n c t i o n s i s common to a l l p o l y s a c c h a r i d e s . Since i t has been demonstrated ( s e c t i o n IIIC) that hydroxyl groups on monosaccharides w i l l r e a ct w i t h c h l o r o t r i a z i n e s , a t t e n t i o n has been d i r e c t e d therefore to 127 Figure I I I - l . S t r u c t u r a l formulae of the polysaccharides sp i n l a b e l l e d v i a cyanuric c h l o r i d e . 128 the r e a c t i o n s of the sp i n l a b e l l i n g reagents [12], [22] and [25] with the hydroxyl groups of polysaccharides. In a r e a c t i o n between a polysaccharide and a d i c h l o r o t r i a z i n e d e r i v a t i v e , i n aqueous media, f i v e p o s s i b l e products can i n p r i n c i p l e be obtained, as shown i n the f o l l o w i n g scheme.* 0 Polysocc X=NH [12 X=0 [22] [44] Aqueous c o n d i t i o n s are d e s i r a b l e s i n c e water s w e l l s and penetrates the polymeric f i b e r s and an a l k a l i n e pH i s required to f a c i l i t a t e the r e a c t i o n Mono-alkoxy or amino c h l o r o t r i a z i n e d e r i v a t i v e s that react r e a d i l y i n the " c o l d " (ambient temperature) such as the s p i n l a b e l reagents [12], [22] and [25], s u f f e r from l o s s by h y d r o l y s i s (degradation to [44]). One s o l u t i o n to t h i s problem i s to s t a b i l i z e the molecule by s u b s t i t u t i n g the second p o s i t i o n w i t h an amino group. However, r e a c t i o n c o n d i t i o n s f o r •adapted from reference 129 the c o u p l i n g r e a c t i o n need be more s t r o n g l y a l k a l i n e and temperatures of over 60°C may be necessary f o r the subsequent r e a c t i o n of a di-amino-t r i a z i n e . This approach has been used s u c c e s s f u l l y i n the dye i n d u s t r y where premixed dyes may be hydrolyzed to a l a r g e degree during long p r i n t -i n g runs. This s t a b i l i z a t i o n procedure was however, not undertaken i n the present study. I t i s i n t e r e s t i n g to note that t h i c k e n i n g agents used i n the d y e s t u f f s i n d u s t r y cannot be based on starches and other r e a c t i v e carbohydrates, but sodium a l g i n a t e ( c o n t a i n i n g only secondary hydroxyl groups) or hydrocarbon emulsions are s a t i s f a c t o r y . This i n d i c a t e s t h a t , at l e a s t w i t h these dye molecules, the r e a c t i o n of the t r i a z i n e moiety i s s e l e c t i v e f o r primary a l c o h o l s of polysaccharides. I t seems remarkable that heterogeneous r e a c t i o n s w i t h i n s o l u b l e polysaccharides such as c e l l u l o s e f i b e r s predominates i n competition w i t h the homogeneous r e a c t i o n w i t h water molecules, even though the l a t t e r are present i n great excess. A p o s s i b l e reason f o r t h i s i s that the molecules which are absorbed by the f i b e r have t h e i r r e a c t i v e groups brought i n t o c l o s e p r o x i m i t y w i t h the f i b e r , making the e f f e c t i v e concen-t r a t i o n of reagent i n the f i b e r much greater than i n the aqueous s o l u t i o n , (b) O p t i m i z a t i o n and Q u a n t i t a t i o n The amount of s p i n l a b e l attached to the polysaccharide depends l a r g e l y upon the r e a c t i o n c o n d i t i o n s used. Aqueous a l k a l i n e r e a c t i o n s proved to be most s u i t a b l e and highest y i e l d s were obtained by the f o l l o w -i n g methods. Water i n s o l u b l e polysaccharides ( c e l l u l o s e , agarose and Sephadex G25) were f i r s t t r e a t e d overnight w i t h 8% sodium hydroxide and the excess sodium hydroxide s o l u t i o n was removed by decantation; to t h i s p r e t r e a t e d m a t e r i a l was then added an aqueous-acetone s o l u t i o n of the t r i a z i n e s p i n l a b e l . A f t e r a s u i t a b l e r e a c t i o n time the product was 130 f i l t e r e d and washed. In c o n t r a s t , water-soluble polysaccharides (guar gum, xanthan gum and starch) were d i s s o l v e d i n 4% sodium hydroxide and to t h i s s o l u t i o n was added an acetone s o l u t i o n of the s p i n l a b e l . The guar and xanthan gums were p r e c i p i t a t e d by adding a l a r g e amount of acetone and then were f i l t e r e d and washed. The s t a r c h sample was p u r i f i e d by g e l f i l t r a t i o n chromatography on Sephadex LH-20. Since the l a b e l l i n g reagents [12], [22] and [25] were i n s o l u b l e i n water t h e i r r e a c t i o n s i n aqueous media were probably l i m i t e d by t h e i r low e f f e c t i v e c o n c e n t r a t i o n . The r e a c t i o n s w i t h the s o l u b l e polysaccharides d i d not have the same advantage as the i n s o l u b l e polysaccharides of e f f e c t i v e l y c o n c e n t r a t i n g the s p i n l a b e l reagents by adsorption and thereby the degree of competing h y d r o l y s i s . Denaturation of agarose, guar and xanthan gums was detected a f t e r t h e i r r e a c t i o n s w i t h the s p i n l a b e l l i n g reagents. Guar and xanthan gums, which normally d i s s o l v e i n water at room temperature were, a f t e r r e a c t i o n , t o t a l l y i n s o l u b l e , even i n b o i l i n g water. Agarose, which normally w i l l d i s s o l v e i n hot water, would no longer d i s s o l v e a f t e r r e a c t i o n . This l a c k of s o l u b i l i t y a f t e r d e r i v a t -i z a t i o n i s most probably due to the formation of c r o s s - l i n k s between the polysaccharide chains introduced by the d i c h l o r o t r i a z i n e s p i n l a b e l s [11] and [12], as shown i n [42]. This i s not an unexpected r e s u l t s i n c e , f o r example dextran, when c r o s s - l i n k e d w i t h e p i c h l o r o h y d r i n (Figure I I I - l ) , forms an i n s o l u b l e d e r i v a t i v e which i s s o l d f o r chromatography under the trade name of Sephadex. I t i s a l s o expected that the NaOH-treatment, e s p e c i a l l y the overnight treatment of c e l l u l o s e and agarose, would cause a c e r t a i n degree of depolymerization of these m a t e r i a l s . Sephadex, on the other hand, has been c r o s s - l i n k e d and i s known to be very s t a b l e to base-treatment. 131 The esr s p e c t r a of these s p i n l a b e l l e d polysaccharides are shown i n Figure I I I - 2 . Although i t i s not the main purpose of t h i s work to analyze i n d e t a i l the l i n e shapes of these s p e c t r a , some important q u a l i t a t i v e i n f o r m a t i o n can be obtained r e a d i l y and w i l l be discussed here.* The m o b i l i t y of the n i t r o x i d e group i s d r a m a t i c a l l y r e f l e c t e d i n the s p e c t r a of Figure I I I - 2 . A r e l a t i v e l y s m a ll n i t r o x i d e molecule such as [10] or [11], tumbling f r e e l y and r a p i d l y i n s o l u t i o n , shows three sharp l i n e s each of equal i n t e n s i t y (see s e c t i o n H I E ) . The spectra i n Figure I I I - 2 are however, very d i f f e r e n t from t h i s and t h e i r broad l i n e s r e f l e c t f o r the most p a r t , the much slower motion of the n i t r o x i d e as a r e s u l t of i t s being a f f i x e d at one end, to a very l a r g e , slow moving macromolecule. The spectrum of l a b e l l e d c e l l u l o s e shows more severe broadening probably due to e l e c t r o n exchange between neigh-bouring n i t r o x i d e s (see H I E ) . As w i l l be discussed i n more d e t a i l , the slower motion of the n i t r o x i d e , broadens the high f i e l d l i n e to a much greater degree than i t does the low f i e l d or center l i n e . Various degrees of motional freedom a l l c o n t r i b u t e to the l i n e shapes of these s p e c t r a . The motion of the polysaccharide backbone and the r o t a t i o n about the bonds j o i n i n g the n i t r o x i d e to the polysaccharide provide most of the motional freedom f o r the appended n i t r o x i d e . These motions, however, depend upon the host environment (solvent) i n which the polysaccharide i s suspended, or *For a more d e t a i l e d d i s c u s s i o n on l i n e shape a n a l y s i s of esr spectra of s p i n l a b e l l e d p o l y s a c c h a r i d e s , the reader i s r e f e r r e d to a report by A p l i n and H a l l 2 0 . 132 1 I Figure III-2. Ambient temperature aqueous esr spectra of (a) cellulose, (b) agarose, (c) Sephadex G-25, (d) guar gum, (e) xanthan gum, and (f) starch, using from l e f t to right, labelling reagents [12], [22] and [25]. 133 d i s s o l v e d . A polar solvent which can penetrate the h y d r o p h i l i c poly-saccharide and s o l v a t e i t , such as water does, imparts much more m o b i l i t y to a pendant n i t r o x i d e than does a non p o l a r organic s o l v e n t . Chloro-form, f o r example, which cannot penetrate the polysaccharide m a t r i x , only a f f e c t s the l a b e l s on the outer e x t r e m i t i e s of the p o l y s a c c h a r i d e , l e a v i n g the inner reaches e f f e c t i v e l y the same as f o r a dry powder. The sp e c t r a i n Figure I I I - 3 of l a b e l l e d Sephadex and agarose i n water and chloroform s o l v e n t s both show t h i s e f f e c t . The esr spectrum i n chloroform does indeed look the same as a t y p i c a l powder spectrum of non i n t e r a c t i n g ( s p i n d i l u t e ) , n i t r o x i d e s (see s e c t i o n H I E ) , where the la r g e s p l i t t i n g 2A„ i s now v i s i b l e . In aqueous media the spectra r e v e a l a much more mobile n i t r o x i d e moiety than f o r the CHCI3 s o l u t i o n and the 2A„ s p l i t t i n g i s now p a r t i a l l y averaged and i s no longer v i s i b l e . I t can be seen from t h i s b r i e f d i s c u s s i o n that s p i n l a b e l l i n g of polysacc-harides can provide considerable i n s i g h t to the way i n which solvents i n t e r a c t w i t h p o l y s a c c h a r i d e s . Determination of the qua n t i t y of s p i n l a b e l s on the surface of these polysaccharides was performed both by elemental a n a l y s i s and by esr double i n t e g r a t i o n of the f i r s t d e r i v a t i v e spectrum w i t h comparison to a standard. These r e s u l t s are tabulated i n t a b l e I I I - l . Unfortun-Figure III-3. Comparison of the aqueous esr spectra of l a b e l l e d Sephadex and Agarose i n (A) with t h e i r chloroform spectra i n (B). 135 TABLE I I I - l : Data obtained from elemental a n a l y s i s and esr double i n t e g r a t i o n of l a b e l l e d polysaccharides mono-saccharide mono-residues saccharide per l a b e l residues from per l a b e l elemental from esr Sample C H N a n a l y s i s i n t e g r a t i o n (a) C e l l u l o s e c a l c d . 44. 44 6. 17 0.0 (b) C e l l u l o s e blank 43. 81 6. 45 0.0 (c) C e l l u l o s e / [ 2 2 ] 43. 16 6. 39 1.18 26 118-2300 (d) C e l l u l o s e / [ 1 2 ] 44. 13 6. 31 0.88 37 (e) C e l l u l o s e + [22] c a l c d . 1.18 (f) C e l l u l o s e + [22] found 44. 07 6. 39 1.03 (g) Agarose c a l c d . 47. ,06 5. ,88 0.0 (h) Agarose blank 46. .40 6. .56 0.0 ( i ) Agarose/[22] 45. .35 6. .24 2.88 10 2900 ( j ) Agarose/[12] 47, .43 6. .73 0.83 40 (k) Sephadex c a l c d . 44 .44 6. .17 0.0 (1) Sephadex blank 37 .43 5 .45 0.0 (m) Sephadex/[22] 45 .31 7 .01 1.59 20 1700 (n) Sephadex/[12] 44 .58 7 .09 0.25 120 900 (o) Guar gum/[22] (P) Xanthan gum/[22] 1900 136 a t e l y both methods s u f f e r from important drawbacks. Elemental a n a l y s i s i s s a t i s f a c t o r y only f o r r e l a t i v e l y high l e v e l s of chemical m o d i f i c a t i o n where the elemental r a t i o s are a l t e r e d s i g n i f i c a n t l y from those of the n a t i v e p o l y s a c c h a r i d e . Since n i t r o g e n i s not present i n the n a t i v e polysaccharides studied here, a n a l y s i s f o r n i t r o g e n o f f e r s a good probe f o r the extent of d e r i v a t i z a t i o n . To check the v a l i d i t y of the elemental a n a l y s i s , a sample ( f ) of c e l l u l o s e was coated non-covalently w i t h a known amount of the l a b e l [22] from a chloroform s o l u t i o n ; i t s a n a l y s i s was the XSI X.NHJ12J X = 0 [22] same as that c a l c u l a t e d ( e ) . Other support f o r the data obtained by elemental a n a l y s i s comes from the f a c t that the "blank," untreated samples showed no dete c t a b l e n i t r o g e n and that carbon and hydrogen a n a l y s i s were ge n e r a l l y acceptable, throughout a l l samples, a f t e r accounting f o r about 3-5% of t i g h t l y bound water. Reactions w i t h the l a b e l l i n g reagent [12] were i n a l l cases l o w e r - y i e l d i n g than the r e a c t i o n s w i t h the alkoxy analogue [22]. This may be accounted f o r by i t s lower s o l u b i l i t y i n aqueous-acetone or more probably by i t s lower r e a c t i v i t y . Should d e r i v -a t i z a t i o n be lower than about one s p i n l a b e l per two hundred saccharide r e s i d u e s , elemental a n a l y s i s i s not s u f f i c i e n t l y s e n s i t i v e to detect t h i s change. F o r t u n a t e l y the esr i n t e g r a t i o n technique i s much more s e n s i t i v e than elemental a n a l y s i s and d e r i v a t i z a t i o n as low as one per s e v e r a l thousand saccharide r e p e a t i n g u n i t s can be detected. In 137 p r i n c i p l e , as long as a f i r s t d e r i v a t i v e spectrum can be obtained, t h i s spectrum can be i n t e g r a t e d to give the t o t a l number of s p i n s , a f t e r comparison w i t h a standard. The f o l l o w i n g equation can be used to c a l c u l a t e the number of spins i n an unknown sample, [STD] A X G S T D [X] = ASTD G X where [X] i s the unknown c o n c e n t r a t i o n , [STD] i s the standard concentra-t i o n of n i t r o x i d e , A i s the area under the absorption mode spectrum measured i n any a r b i t r a r y u n i t as long as they are the same f o r both standard and unknown, and G i s the r e l a t i v e gain of the spectrum ampli-f i e r . For t h i s equation, the microwave power and modulation amplitude must be kept the same f o r both the standard and unknown. This technique, however, p o s s i b l y due to i t s high s e n s i t i v i t y , gave unreproducible r e s u l t s (see Table I I I - l ) and q u i t e d i f f e r e n t from those obtained from elemental a n a l y s i s . 21 This technique i s known to be subject to many sources of e r r o r s . One p o t e n t i a l l y l a r g e source a r i s e s from the f a c t that an esr probe c a v i t y i s only s e n s i t i v e over a very small area. Hence, the t o t a l weight of the sample i n the c a v i t y cannot be used to c a l c u l a t e the number of spins per gram. Instead, the s o l i d samples have to be treated as " c o n c e n t r a t i o n s " and l a r g e e r r o r s may have r e s u l t e d from uneven packing d e n s i t i e s . In a d d i t i o n , the second i n t e g r a t i o n was c a r r i e d out by means of peak c u t t i n g i n the presence of poor b a s e l i n e s . In g e n e r a l , p o t e n t i a l l y l a r g e sources of e r r o r s can come from f a i l u r e to s a t i s f y the f o l l o w i n g c o n d i t i o n s . 1. The same solvent or host and the same sample geometry should be employed to ensure that the microwave magnetic f i e l d Hj i s the 138 same f o r the unknown and the standard samples. 2. There should be no s a t u r a t i o n f o r e i t h e r sample or standard and the microwave power l e v e l should be the same f o r both sample and standard. 3. The unknown and standard should be at the same temperature. 4. The unknown and standard sample tubes must be i n e x a c t l y the same p o s i t i o n s i n the esr c a v i t y . 5. The standard and unknown should have a s i m i l a r l i n e shape and c o n c e n t r a t i o n . While c o n d i t i o n s 2 and 3 are e a s i l y met, 1, 4 and 5 are not. A n i t r o x i d e i n a s o l i d p o lysaccharide sample cannot be considered to be i n the same host as the s o l u t i o n standard even i f both samples are run i n the same l i q u i d . Since the instrument used i n t h i s study d i d not have a dual c a v i t y , the sample and standard could not be run simultaneously and equivalent sample p o s i t i o n s were d i f f i c u l t to achieve. The l i n e shapes of both standard and sample were a l s o q u i t e d i f f e r e n t . The polysacchar-ide samples had very broad esr l i n e widths i n comparison to the very narrow l i n e s of the s o l u t i o n standard. This technique, however, even 22 w i t h i t s drawbacks, has been used s u c c e s s f u l l y by others and i t should be p o s s i b l e to o b t a i n at l e a s t an accuracy of ±50%. Since the r e s u l t s reported here sometimes c a r r i e d a much higher d e v i a t i o n than t h i s , even between runs on the same sample, i t i s f e l t i n r e t r o s p e c t that the performance of the instrument used i n t h i s study was simply not compat-i b l e w i t h these experiments. (c) Evidence f o r a Covalent Bond One of the assumptions i n t h i s work i s that a covalent bond e x i s t s that j o i n s the t r i a z i n e moiety to the polysaccharide m a t r i x , and that the 139 t r i a z i n e u n i t i s not bound by some other non-covalent a s s o c i a t i o n or by entrapment. This i s very d i f f i c u l t to prove d i r e c t l y but some evidence f o r the chemical union between r e a c t i v e dyes and c e l l u l o s e i s convincing and i s summarized below:* 1. I f cotton i s dyed w i t h a P r o c i o n M dye (trade name f o r a c h l o r o -t r i a z i n e dye) from a n e u t r a l bath and then washed w i t h water, most of the c o l o r i s removed; i f , however, the dyed f a b r i c i s treated w i t h a l k a l i most of the dye becomes f i x e d and i s not even removed i n a b o i l i n g soap bath. 2. P r o c i o n dyes are not removed from dyed cotton by e x t r a c t i o n w i t h b o i l i n g p y r i d i n e , O-chlorophenol or chloroform. On the other hand d i r e c t v a t - and azoic-dyes are s t r i p p e d by these s o l v e n t s . 3. C e l l u l o s e dyed w i t h Procion M dyes are i n s o l u b l e i n cuprammonium s o l u t i o n s , but s i m i l a r f i b e r dyed w i t h d i r e c t dyes d i s s o l v e completely. 4. When cotton dyed w i t h the monoazo dye P r o c i o n y e l l o w M-R, i s t r e a t e d w i t h sodium h y d r o s u l p h i t e , the dye i s s p l i t i n t o two components. The component c o n t a i n i n g the r e a c t i v e group remains anchored to the f i b e r and can be d i a z o t i z e d and coupled w i t h an amine or phenol to give a new dye. In t h i s t h e s i s work some evidence f o r the probable occurrence of a t t a c h -ment comes from the f a c t that monosaccharide t r i a z i n e s p i n l a b e l compounds can be synthesized as already shown i n I I I B ( i ) and ( i i ) . Further i n f o r m a t i o n concerning the bond between the c h l o r o t r i a z i n e d e r i v a t i v e s and polysaccharides was obtained by esr spectroscopy; the appropriate esr spectra o f f e r i n g t h i s a d d i t i o n a l i n f o r m a t i o n are shown 4 *from reference . 140 i n Figure I I I - 4 . I f c e l l u l o s e , agarose or Sephadex are t r e a t e d w i t h the s p i n l a b e l l i n g agent C&2T0S& [22] under n e u t r a l c o n d i t i o n s , there i s no d e t e c t a b l e s i g n a l a f t e r washing with aqueous-acetone (A). However, i f the polysaccharide i s f i r s t t r e a t e d w i t h NaOH and then [22] added, the r e s u l t i n g h i g h l y "immobile" esr s i g n a l i n (B) cannot be removed by prolonged washing. To e l i m i n a t e the hydroxide as having any independent e f f e c t , the "non r e a c t i v e " s p i n l a b e l H0S£ [11] was added to the a l k a l i t r e a t e d polysaccharides i n an i d e n t i c a l procedure to that used f o r the C£2T0S£ r e a c t i o n s , and the product washed. A f t e r b r i e f water washings the esr s p e c t r a i n (C) were obtained. Upon more thorough washing the s i g n a l s were completely removed. Thus the s p e c t r a i n (C) show that esr spectroscopy can be used to d i s t i n g u i s h between "bound" and " f r e e " n i t r o x i d e s w i t h i n the polysaccharides. These " f r e e " s i g n a l s come from m o t i o n a l l y r e s t r i c t e d populations of n i t r o x i d e s temporarily trapped w i t h i n aqueous voids i n the p o l y s a c c h a r i d e s , which are l a r g e enough to allow unhindered m o b i l i t y . Given s u f f i c i e n t time, these unbound n i t r o x i d e s can d i f f u s e out and be washed away. While these " f r e e " s i g n a l s i n (C) provide proof of a non c o v a l e n t l y bound n i t r o x i d e , the h i g h l y immobile spectrum of (B) does not alone prove the existence of a covalent bond since i t does not e l i m i n a t e the p o s s i b i l i t y of t i g h t l y adsorbed, or trapped l a b e l s . The combination of evidence from (A) and (B) however, would seem to r u l e out t h i s p o s s i b i l i t y , (d) Distance Measurements A very u s e f u l piece of i n f o r m a t i o n that can be extracted from many esr s p e c t r a of l a b e l l e d m a t e r i a l s i s the distance between adjacent p a i r s of free r a d i c a l s . The parameter dj/ d shown i n Figure I I I - 5 was f i r s t 23 used by Kokorin et a l . on an e m p i r i c a l b a s i s to c h a r a c t e r i z e i n t e r -CELLULOSE AGAROSE SEPHADEX Figure TII-4. C o n t r o l esr experiments where (A) shows the esr spectra of the polysaccharides l a b e l l e d w i t h [22] under n e u t r a l c o n d i t i o n s , (B) shows the "bound" esr spectra of the polysaccharides l a b e l l e d with [22] under basic, con-d i t i o n s and (C) shows the " f r e e " esr spectra of the three polysaccharides l a b e l l e d w i t h the non-re.ictive s p i n l.ibc) [11] HOSP, under bnsic c o n d i t i o n s with b r i e f wnshing. Figure I I I - 5 . Powder spectrum showing the heights d 1 and d and the s p l i t t i n g 2A. 143 act i o n s between n i t r o x i d e s . I t has r e c e n t l y been shown i n our l a b o r a -22 tory by J . D. A p l i n and J . C. Waterton t h a t , dl _ -3 + 0.58 r ^ d i l where r i s the mean nearest neighbour d i s t a n c e and (di/d) , . n i s the d i / d value f o r the l a b e l l e d m a t e r i a l at i n f i n i t e d i l u t i o n (spins f a r enough apart so that no d i p o l a r i n t e r a c t i o n s can occur between neighbouring n i t r o x i d e s ) . This value i s u s u a l l y about 0.4 but i s dependent upon the m a t e r i a l and solvent-host used and th e r e f o r e i t should be checked f o r each system. The di s t a n c e values obtained f o r s p i n l a b e l l e d c e l l u l o s e , agarose and Sephadex are shown i n Table I I I - 2 . The distance r can al s o TABLE I I I - 2 : Distance measurements^ determined f o r polysaccharides l a b e l l e d w i t h [22] 7C Sample (dj/d) ^ dj/d rnm C e l l u l o s e 0.42 0.66 1.33 Agarose 0.48 2.11 Sephadex - 0.42 -^measured at 77K i n chloroform *a s p i n d i l u t e l a b e l l e d c e l l u l o s e sample was used as (di/d) .... f o r a l l samples 1 °°dil be used to i n f e r r e l a t i v e l e v e l s of l a b e l l i n g between samples of the same m a t e r i a l but only i f the l a b e l l i n g i s purely random and no c l u s t e r s of l a b e l s e x i s t . Distance i n f o r m a t i o n i s u s e f u l i n determining the surface topography of polymeric and macromolecular m a t e r i a l s and may be e s p e c i a l l y u s e f u l i n chromatography and membrane s t u d i e s . This technique has been used i n 144 t h i s l a b o r a t o r y , by o t h e r s , t o : determine the outside and i n s i d e d i a -meter of s p h e r i c a l liposome v e s i c l e s (model membranes); to study the d i s t r i b u t i o n of n i t r o x i d e s layered on a s i l i c a surface [ I H C ( i v ) ] and to 22 determine the " a c c e s s i b l e " surface area of c e l l u l o s e ( i i ) Bovine Serum Albumin The amino groups of a v a r i e t y of p r o t e i n s are a l s o amenable to m o d i f i c a t i o n v i a the chemistry of cyanuric c h l o r i d e . Spin l a b e l l i n g of 19 15 p r o t e i n s i s not new and L i k h t e n s h t e i n et a l . have used the t r i a z i n e s p i n l a b e l reagent [12] to l a b e l the l y s i n e e amino groups of p r o t e i n s i n the hope of i n v e s t i g a t i n g t h e i r s t r u c t u r e and f u n c t i o n . In the present work the new s p i n l a b e l l i n g reagent [22] was used to l a b e l bovine serum albumin (BSA), the esr spectrum of which i s shown i n Figure I I I - 6 . BSA i s a globular plasma p r o t e i n , molecular weight approximately 67,000 which contains 55 l y s i n e residues. From esr double i n t e g r a t i o n i t was shown that the number of l y s i n e residues l a b e l l e d w i t h t h i s reagent i s about 12 per p r o t e i n molecule. Although a d i s t a n c e measure-ment was not done, the spectrum i n Figure I I I - 6 i n d i c a t e s a degree of e l e c t r o n exchange between neighbouring n i t r o x i d e s . This t h e r e f o r e i m p l i e s that some of the s p i n l a b e l s are i n a c l o s e proximity (<_ 1 nm). This l a b e l l i n g reagent [22] has an advantage over i t s amino s u b s t i t u t e d cousin [12] i n that i t can be reacted under much milder c o n d i t i o n s which are more s u i t a b l e f o r p r o t e i n s , ( i i i ) Aluminum Oxide The surface hydroxyl groups of aluminum oxide AJI2O3 (alumina) are a l s o s u s c e p t i b l e to r e a c t i o n w i t h c h l o r o t r i a z i n e d e r i v a t i v e s , and to f u r t h e r i l l u s t r a t e again that the chemistry of cyanuric c h l o r i d e can be a p p l i e d to other areas as w e l l as p o l y s a c c h a r i d e s , alumina was l a b e l l e d 145 Figure I I I - 6 . Ambient temperature aqueous esr spectrum of BSA l a b e l l e d w i t h reagent [22]. 146 w i t h [22]. In f a c t t h i s experiment was performed, purely by accident, when the above reagent [22] was being chromatographed on alumina with chloroform i n a t e s t to see i f unreacted cyanuric c h l o r i d e could be separated from an alkoxy s u b s t i t u t e d t r i a z i n e d e r i v a t i v e . I t was found that compound [22] became bound by the column and could not be e l u t e d . This i n i t i a l observation was subsequently followed up w i t h c o n t r o l l e d experiments. A 1:1 mixture, by weight, of alumina (Basic Brockman a c t i v i t y 1) and [22] i n chloroform was reacted, f i l t e r e d and then washed w i t h methanol and chloroform to remove any unbound m a t e r i a l s . The esr spectrum of t h i s m a t e r i a l i s shown i n Figure I I I - 7 ( i ) . 2 0 G Figure I I I - 7 ( i ) . Ambient temperature chloroform esr spectrum o alumina l a b e l l e d w i t h [22]. 147 This spectrum i s again c h a r a c t e r i s t i c of a h i g h l y immobilized spin l a b e l w i t h p o s s i b l y some degree of exchange broadening. As before, esr double i n t e g r a t i o n and elemental a n a l y s i s were used to q u a n t i t a t e the extent of l a b e l l i n g (Table I I I - 3 ) . As i n the polysaccharide work, TABLE II1 - 3 : Extent- of d e r i v a t i z a t i o n of A £ 2 0 3 w i t h reagent [22] Sample Spins per gram from elemental a n a l y s i s H N Spins per gram from esr double i n t e g r a t i o n (a) AJI2O3 c a l c d . 0.0 - 0.0 (b) AH2^3 blank 0.03 0.45 0.0 (c) AJI2O3 + [22] c a l c d . 0.80 - 0.31 (d) A£ 2°3 + [22] f o u n d 1.24 0.77 0.24 19 1 8 (e) A£ 20 3/[22] 0.50 0.57 0.20 2.1 x 10 9.0 x 10 there was a discrepancy between these two techniques although the agree-ment here i s acceptable. The sample f o r (c) and (d) was run as a con-t r o l where a known amount of A£203 and [22], were mixed i n chloroform and the mixture evaporated to dryness. The a n a l y s i s on t h i s m a t e r i a l i s acceptable and even t h i s low n i t r o g e n percentage could be detected w i t h reasonable accuracy. The d i f f e r e n c e of about 60% between the elemental a n a l y s i s and i n t e g r a t i o n values i s w i t h i n experimental e r r o r since an u n c e r t a i n t y of about ±50%. i s expected w i t h the i n t e g r a t i o n and about ±30% f o r the elemental a n a l y s i s . A c o n t r o l experiment was a l s o performed to support the c l a i m of covalent bonding between [22] and the A2.2O3 surface. The n i t r o x i d e H0S£ [11] was added to alumina i n the same way as C£2T0S£ [22] had been and t h i s sample was then f i l t e r e d and washed overnight w i t h methanol 148 and chloroform. A f t e r t h i s washing, a s m a l l amount of l a b e l remained and i t s esr spectrum, shown i n Figure I I I - 7 ( i i ) (B^,'was c h a r a c t e r i s t i c of a h i g h l y immobilized n i t r o x i d e . This i n d i c a t e s that there i s e i t h e r a strong non covalent bond formed between the surface and the n i t r o x i d e or that these n i t r o x i d e molecules are trapped i n very small pores which r e s t r i c t t h e i r motion. However, assuming that l i n e widths of the [22] t r e a t e d alumina (A) and of the c o n t r o l spectrum of (B) are roughly the same and that sample volumes are the same (a good assumption since alumina packs evenly and the whole c a v i t y was f i l l e d i n both cases), then comparison of r e c e i v e r gain values shows that the alumina l a b e l l e d w i t h C£2T°S£ [22] contains almost 250 times more spins than the sample tr e a t e d w i t h H0S& [11]. Even though these two molecules do not have the same s t r u c t u r e s and an equivalent adsorption f a c t o r cannot be assumed, t h i s r e s u l t s t i l l gives strong evidence that a covalent l i n k a g e i s formed i n the r e a c t i o n of [22] and alumina. This bond can be i l l u s t r a t e d i n two p o s s i b l e ways as shown i n the f o l l o w i n g schematic. 149 Figure I I I - 7 ( i i ) . Ambient temperature chloroform esr spectra of (A) alumina l a b e l l e d w i t h [22] (same as Figure I I I - 7 ( i ) ) and (B) alumina l a b e l l e d w i t h the non-reactive reagent [11] HOS£ as a c o n t r o l . 150 The s t r u c t u r e represented i n (B) i s supported p a r t i a l l y by the f o l l o w i n g experiments. When cyanuric c h l o r i d e was f i r s t reacted w i t h alumina and then CJ^TOSi [22] was added, no l a b e l was incorporated. I t therefore seems that cyanuric c h l o r i d e e f f e c t i v e l y blocks a l l of the r e a c t i v e " b i n d i n g " s i t e s . When the bound cyanuric c h l o r i d e was subsequently reacted w i t h an a l k a l i mixture of H0S£ [11], no esr s i g n a l was detected a f t e r washing. This can e i t h e r mean that s t r u c t u r e (B) i s c o r r e c t or that simply, the remaining c h l o r i n e s are hydrolyzed before or during the attempted r e a c t i o n w i t h H0S£ [11]. Again, as i n the case of the po l y s a c c h a r i d e s , a di s t a n c e between n i t r o x i d e s could be c a l c u l a t e d from the 77K esr spectrum of the CJlpTOSJi [22] l a b e l l e d alumina. To obt a i n an i n f i n i t e d i l u t i o n (di/d) ... °°dil val u e , a s e r i e s of s p i n d i l u t i o n experiments were performed. By r e a c t -i n g samples of alumina w i t h e i t h e r a 1:1.7 or a 1:4 mole r a t i o of [22]/cyanuric c h l o r i d e ) the spin density was s u c c e s s i v e l y d i l u t e d . The comparison of these spectra w i t h the un d i l u t e d case i s shown i n Figure I I I - 8 . Since alumina packs evenly i n CHC&3 these s p e c t r a represent the same amount of sample w i t h i n the esr c a v i t y . R e l a t i v e r e c e i v e r gain and s i g n a l to noise values can be used as a comparison of l e v e l s of spin l a b e l l i n g . The dj/d value f o r the 1:1.7 d i l u t i o n gave a value of 0.43 and was used as the i n f i n i t e d i l u t i o n v a l u e . The dj/d value f o r the u n d i l u t e d sample was 0.739 and the distance r c a l c u l a t e d [as i n I I I C ( i ) ( d ) ] to be 1.24 nm. The 77K spectra of these two samples are shown i n Figure I I I - 9 . ( i v ) C o n t r o l l e d Pore Glass In the same fa s h i o n as A2.203* Si02 can a l s o be spin l a b e l l e d w i t h C£2T0S£ [22]. Dr. J . C. Waterton, w h i l e at UBC, reacted [22] w i t h 151 Figure I I I - 8 . Ambient temperature chloroform esr spectra of (A) alumi l a b e l l e d w i t h [22] (same as Figure I I I - 7 ( i ) ) , Receiver Gain (2 x 10 ), (B) alumina l a b e l l e d w i t h a 1:1.7 molar r a t i o of [22] to cyanuric c h l o r i d e , Receiver Gain (3.2 x 10 4) , and (C) alumina l a b e l l e d w i t h a 1 molar r a t i o of [22] to cyanuric c h l o r i d e , Receiver Gain (5 * 10 ). 152 Figure I I I - 9 . 77K frozen chloroform esr spectra of (A) alumina l a b e l l e d w i t h u n d i l u t e d [22] and (B) alumina l a b e l l e d w i t h a 1:1.7 molar r a t i o of [22] to cyanuric c h l o r i d e . 153 22 modified c o n t r o l l e d pore glass beads as shown below . F i r s t p r o p y l -amine groups were bonded to the surface by at l e a s t one s i l o x a n e bond. 0 4 OH + ( E T O ) 3 S i ( C H 3 ) 3 N H 2 h-OH S i ^ N H z Then the s i l i c a bound amino groups was reacted w i t h C£2TOS£ [22] to give the s p i n l a b e l l e d glass surface. The amount of propylamine groups attached to the surface was determined by elemental a n a l y s i s to be 20 20 6 x 10. ligands/gram. Of these about 1 * 10 amines were reacted w i t h l a b e l [22], as determined by esr double i n t e g r a t i o n , to give an 18% y i e l d . The esr spectrum of t h i s l a b e l l e d s i l i c a i s shown i n F i g 111-10. In h i s work, Dr. Waterton s p i n l a b e l l e d the propylamino modified g l a s s w i t h a v a r i e t y of other c h e m i s t r i e s to develop techniq f o r studying modified s u r f a c e s . 154 Figure 111-10. Ambient temperature methanol esr spectrum of s i l i c a ( c o n t r o l l e d pore glass modified w i t h propylamine groups) l a b e l l e d w i t h reagent [22]. (v) Summary and Conclusions Several p o l y s a c c h a r i d e s , c e l l u l o s e , agarose, Sephadex G-25, guar gum, xanthan gum, and s t a r c h , have been s p i n l a b e l l e d v i a the " t r i v a l e n t " c o u p l i n g reagent, cyanuric c h l o r i d e . I t was found that aqueous, a l k a l i n e c o n d i t i o n s were s u i t a b l e f o r the coupling procedure and that a l k a l i - p r e t r e a t m e n t of the i n s o l u b l e polysaccharides before a d d i t i o n of the s p i n l a b e l , gave the highest y i e l d s . C o n s i s t e n t l y , the oxygen l i n k e d s p i n l a b e l t r i a z i n e compound gave higher y i e l d s than d i d the ni t r o g e n l i n k e d analogue. From the esr spe c t r a of these n i t r o x i d e l a b e l l e d p o l y s a c c h a r i d e s , a wealth of i n f o r m a t i o n concerning both the r e a c t i o n i t s e l f and the r e s u l t -ing labelled m a t e r i a l s can be obtained. This i n f o r m a t i o n i n c l u d e s : the 155 q u a n t i t y of l a b e l attached; the m o b i l i t y of the surface bound n i t r o x i d e ; the e f f e c t s of s o l v e n t s on the environment of the polysaccharide and l a b e l ; the d i f f e r e n c e between t i g h t l y bound and unbound " f r e e " n i t r o x i d e molecules; evidence f o r covalent bonding between the polysaccharide and the t r i a z i n e l o c u s ; and the average mean nearest neighbour distance between n i t r o x i d e l a b e l l e d s i t e s . The q u a n t i t a t i o n of l a b e l s , by the esr double i n t e g r a t i o n technique was found to give unacceptable r e s u l t s and instead elmental m i c r o a n a l y s i s was used. This technique gave rep r o d u c i b l e r e s u l t s and c o n t r o l samples were used to check i t s v a l i d i t y . The number of monosaccharide u n i t s between each t r i a z i n e s p i n l a b e l group, f o r the three i n s o l u b l e p o l y -s a c c h a r i d e s , ( c e l l u l o s e , agarose, and Sephadex G-25) l a b e l l e d w i t h C&2TOS2, [22], were 26, 10, and 20 u n i t s r e s p e c t i v e l y . L a b e l l i n g w i t h the n i t r o g e n analogue C£2TNHS£ [12] gave l e v e l s of 37, 40, and 120 u n i t s r e s p e c t i v e l y . From the l i n e shapes of the esr spectra i t was shown that water penetrates these polysaccharides to s o l v a t e the polysaccharide f i b e r s and the appended n i t r o x i d e , whereas chloroform does not penetrate and leaves the polysaccharide e s s e n t i a l l y as i f i t were a dry powder. The esr s p e c t r a of these l a b e l l e d p o l y s a c c h a r i d e s , along w i t h v a r i o u s c o n t r o l experiments, gave strong evidence supporting the e x i s -tence of a covalent bond and a l s o could be used to d i s t i n g u i s h between bound and unbound " f r e e " n i t r o x i d e m o i e t i e s . This l a t t e r i n f o r m a t i o n can be used to determine c o r r e c t washing procedures to remove unreacted m a t e r i a l s and i s of s u b s t a n t i a l p r a c t i c a l importance f o r the t e x t i l e i n d u s t r y , f o r example. The average mean distance r between neighbouring n i t r o x i d e s was 156 found to be 1.3 ran i n l a b e l l e d c e l l u l o s e and 2.1 nm f o r l a b e l l e d agarose. The distance i n l a b e l l e d Sephadex must have been l a r g e r than 2.5 nm s i n c e no d i p o l a r i n t e r a c t i o n was detected. The f a c t that the l a b e l s on c e l l u l o s e are c l o s e r together than i n the other two polysaccharides and yet out of these three, the q u a n t i t y of l a b e l s on c e l l u l o s e was the lowest, i s a r e f l e c t i o n of the smaller a c c e s s i b l e surface area of 24 c e l l u l o s e due to i t s many h i g h l y c r y s t a l l i n e regions Bovine serum albumin (BSA), aluminum oxide, and c o n t r o l l e d pore g l a s s were al s o d e r i v a t i z e d w i t h cyanuric c h l o r i d e . About 12 out of 55 a v a i l a b l e e - l y s i n e residues of BSA were l a b e l l e d w i t h the s p i n l a b e l l i n g reagent. C J I 2 T O S X , [22]. Aluminum oxide was a l s o l a b e l l e d w i t h t h i s same reagent to the extent of about 2 x 1 0 1 9 spins per gram w i t h a mean average d i s t a n c e between spins of 1.24 nm. Strong evidence, f o r a c o n t r o l experiment a l s o obtained from esr spectroscopy, supported the existence of a co-v a l e n t bond between the alumina surface hydroxyl groups and the t r i a z i n e moiety. •A chemically modified s i l i c a surface (propylamine modified c o n t r o l l e d pore glass) was a l s o l a b e l l e d w i t h the same s p i n l a b e l t r i a z i n e at a l e v e l of about 1 * 1 0 2 0 spins per gram. We suggest that the use of s p i n l a b e l l i n g v i a cyanuric c h l o r i d e o f f e r s a powerful t e s t v e h i c l e f o r the f u t u r e m o d i f i c a t i o n of these and other s u b s t r a t e s . Information such as optimum r e a c t i o n c o n d i t i o n s , d i s t a n c e s , solvent s u b s t r a t e i n t e r a c t i o n s , evidence f o r covalent bonds, e t c . , can then be a p p l i e d more g e n e r a l l y to the understanding of other t r i a z i n e mediated surface m o d i f i c a t i o n s whose products might otherwise be d i f f i c u l t to c h a r a c t e r i z e . H I D : Nmr Spectroscopy The nmr spectroscopy i n t h i s s e c t i o n i s r e s t r i c t e d to the spec t r a obtained from some of the compounds synthesized i n s e c t i o n I I I B and C. No background to the technique w i l l be given here because nmr spectr o -scopy i s now used so r o u t i n e l y as a s t r u c t u r a l t o o l by organic and in o r g a n i c chemists. Normal *H nmr spec t r a can be obtained f o r the diamagnetic compounds of s e c t i o n I I I B and C as shown i n the two example spec t r a of compounds [21] and [32] i n Figure I I I - l l and Figure 111-12 r e s p e c t i v e l y . The protons i n the three sugar r i n g s of [32] (Figure 111-12) are a l l equiv-a l e n t because of the C3 symmetry a x i s which e x i s t s through the center of the t r i a z i n e r i n g . The m a j o r i t y of the compounds i n I I I B ( i ) and ( i i ) are, however, paramagnetic s i n c e they c o n t a i n the f r e e r a d i c a l n i t r o x i d e group. This broadens the l i n e s i n the proton spectrum considerably and makes a s s i g n -ments and hence s t r u c t u r a l i n f o r m a t i o n d i f f i c u l t to e x t r a c t . This problem can simply be overcome though, by reducing the n i t r o x i d e w i t h sodium h y d r o s u l f i t e ( d i t h i o n i t e N a ^ O ^ • 2H 20) . Other reducing agents besides d i t h i o n i t e can be u s e d " and a l s o , t h i s step can be reversed by r e o x i d i z i n g 2 5 the reduced l a b e l . To i l l u s t r a t e t h i s r e d u c t i o n technique the spectrum of compound [36] i s shown i n Figure 111-13, before and a f t e r 158 Figure I I I - l l . Proton nmr spectrum (270 MHz) of compound [21] in deuterioacetone. Figure 111-12. Proton nmr spectrum (270 MHz) of compound [32]. 160 Figure 111-13. Proton nmr spectrum (270 MHz) of compound [36] before and a f t e r r e d u c t i o n . 161 r e d u c t i o n w i t h d i t h i o n i t e . The r e s u l t i n g nmr spectrum of t h i s compound i s indeed very i n t e r e s t i n g . There does not e x i s t a proper symmetry element i n t h i s molecule and i t i s therefore s u r p r i s i n g that both sugar r i n g s appear equivalent i n the spectrum. The problem here i s the same as that encountered e a r l i e r f o r the ferrocene analogue i n chapter I I . I f the bond j o i n i n g the oxygen atom of the n i t r o x i d e to the t r i a z i n e r i n g i s able to r o t a t e f r e e l y ( f a s t on the nmr time s c a l e ) then there can be s a i d to e x i s t a pseudo C 2 a x i s of symmetry which can make the two sugar m o i e t i e s e f f e c t i v e l y e q u i v a l e n t . This n i t r o x i d e group i s rather small i n s i z e and innocuous i n e l e c t r o n i c nature i n comparison to the aromatic ferrocene group of compound [37]. Due to t h i s , the n i t r o x i d e group would not be expected to create as l a r g e a chemical s h i f t d i f f e r e n t i a l between the two sugar r i n g s and t h e r e f o r e a slower bond r o t a t i o n i s r e q u i r e d to create the pseudo C 2 symmetry a x i s needed to make the protons of the two sugar r i n g s appear e q u i v a l e n t . SGIc OvN To prepare the reduced samples, the compound was f i r s t d i s s o l v e d i n d e u t e r i o c h l o r o f o r m and then washed w i t h aqueous sodium d i t h i o n i t e . As the n i t r o x i d e became reduced i t s orange c o l o r disappeared, however, due to r a p i d decomposition of the d i t h i o n i t e i n water, t h i s washing must be repeated a few times. The deuteriochloroform l a y e r was then 162 separated and d r i e d over sodium s u l f a t e before i t s nmr spectrum was run. H I E : Esr Spectroscopy of N i t r o x i d e s The f o l l o w i n g s e c t i o n has been adapted, i n p a r t , from a Ph.D. 26 t h e s i s w r i t t e n by Dr. J . D. A p l i n , and a B.Sc. t h e s i s w r i t t e n by A r t „ 16 van der Est E l e c t r o n s p i n resonance (esr) occurs i n a paramagnetic molecule when t r a n s i t i o n s between the Zeeman l e v e l s , whose degeneracy may be l i f t e d by the a p p l i c a t i o n of a magnetic f i e l d H, are induced by an electromagnetic f i e l d Hi of frequency v. The separation of these Zeeman l e v e l s i s described i n hv = gBH (1) where h i s Plank's constant, B the Bohr magneton (eh/2m), m the mass of the e l e c t r o n , and g a dimensionless parameter r e l a t e d to the e f f e c t i v e magnetic moment of the e l e c t r o n (jJ^) by = -gBS (2) where Sh i s the s p i n angular momentum ve c t o r . D i f f e r e n c e s i n the Zeeman energy between d i f f e r e n t molecules r e s u l t i n changes i n g from i t s f r e e e l e c t r o n spin-only value of 2.00232, as a r e s u l t of spin o r b i t coupling. Thus the g value i s used to c h a r a c t e r i s e the p o s i t i o n of the resonance i n the frequency spectrum. The s p i n Hamiltonian f o r a n i t r o x i d e e l e c t r o n i s given by <%=*jt (Zeeman) +*# (hyperfine) ( d i p o l a r ) +«# (exchange) = 3\H«g-S_ + S/T-I + JS-D-jS + J'S/S (3) where ^ and I_ are the e l e c t r o n s and nuclear s p i n operators r e s p e c t i v e l y , and the nuclear Zeeman term has been omitted. The e l e c t r o n g-value, and A, the e l e c t r o n - n u c l e a r h y p e r f i n e c o u p l i n g , are required to be expressed 163 as second-rank tensors because they represent direction-dependent quan-t i t i e s which i n t u r n r e f l e c t the symmetry of the molecule. The h y p e r f i n e c o u p l i n g constant c o n s i s t s of a contact term a 0 I - S (4) where the i s o t r o p i c c o u p l i n g constant ag i s a s c a l a r defined by a 0 - ^ g B g ^ k W l 2 (5) and i>(0) i s the unpaired e l e c t r o n wave f u n c t i o n at the nucleus: and a d i p o l a r i n t e r a c t i o n given by (.I-jOr 2 - 3 ( I - r ) ( S - r ) I T ' S = -J (6) where r i s the electron-nucleus distance v e c t o r . While the contact term r e q u i r e s S - o r b i t a l character i n <Kr) ( | iJj(O) | 2 1 0 ) , the d i p o l a r term d i s -appears i f the e l e c t r o n d i s t r i b u t i o n has s p h e r i c a l symmetry. In the present case, as shown below,* the p r i n c i p a l hyperfine i n t e r a c t i o n occurs between the e l e c t r o n , shown i n the n i t r o g e n 2p z o r b i t a l , and the n i t r o g e n leus ( I = 1). The t r a n s i t i o n s obey s e l e c t i o n r u l e s An^ = 0, Amg = ±1 nuc •adapted from r e f . 21. 164 as shown i n Figure 111-14. The direction-dependence-of the Zeeman and hyperfine i n t e r a c t i o n s may most c l e a r l y be demonstrated experimentally by the esr spectrum of a 27 diamagnetic host c r y s t a l "doped" w i t h n i t r o x i d e (Figure 111-15) Both the p o s i t i o n g and the s p l i t t i n g of the l i n e s A are d i r e c t i o n -dependent g i v i n g r i s e to the p r i n c i p a l values g , g , g ; A , A , f a b r r- °xx yy zz XX yy A2 Z - As might be expected from the molecular symmetry, both tensors are approximately a x i a l l y symmetric, that i s , g ^ 2 (7) B x x — & y y T 5 z z and A ^ A 4 A (8) xx — yy zz These are sometimes given formal a x i a l symmetry by the n o t a t i o n g = 2 = e, (9) S x x 8 y y B± A = A = A, (10) xx yy A = A,, (12) zz The spectrum obtained from a d i l u t e s o l u t i o n of n i t r o x i d e a l s o con-t a i n s three sharp l i n e s (Figure 111-16), but here the g and A a n i s o t r o p i e s (g„, g, and A„, A^) have been averaged so that only the i s o t r o p i c s p l i t -t i n g constant ao remains. Figure 111-16 a l s o shows the spectrum of a p o l y c r y s t a l l i n e array of n i t r o x i d e s , as might be obtained by r a p i d - f r e e z i n g a d i l u t e s o l u t i o n i n t o a g l a s s . A l l p o s s i b l e o r i e n t a t i o n s of the n i t r o x -ide c o n t r i b u t e to the spectrum, which i s simply the sum of resonances due to the o r i e n t a t i o n s shown i n Figure 111-15 together w i t h a l l others. I t i s c l e a r , t h e r e f o r e , that while the c e n t r a l maxima contains c o n t r i b u t i o n s from a l l o r i e n t a t i o n s , the outer extrema of the spectrum are due to r a d i c a l s o r i e n t e d w i t h the molecular z-axis p a r a l l e l to the e x t e r n a l f i e l d , 165 m s rrij 1 AE = hv Figure 111-14. Energy l e v e l diagram f o r a n i t r o x i d e (S = i ) i n a magnetic f i e l d w i t h h y p e r f i n e c o u p l i n g to the s p i n 1 n i t r o g e n nucleus (adapted from reference (21) Swartz, Bolton & Borg). 166 Figure 111-15. D i r e c t i o n a l dependence of Zeeman and hyperfine i n t e r a c -t i o n s demonstrated by the spectrum of a diamagnetic host c r y s t a l "doped" w i t h n i t r o x i d e (adapted from reference (27)). 167 Figure 111-16. Esr s p e c t r a of m a g n e t i c a l l y d i l u t e d i - t - b u t y l n i t r o x i d e (a) i n a p o l y c r y s t a l l i n e s o l i d , (b) a viscous s o l u t i o n , and (c) a non-viscous s o l u t i o n showing g- and A - a n i s o t r o p i e s (adapted from reference (21) Wertz & B o l t o n ) . 168 and that at the " r i g i d l i m i t " the s p l i t t i n g between gives a measure of 2A„. Between the two extremes of non-viscous s o l u t i o n (where T ^ l O ^ s c f o r a s m a l l molecule) and p o l y c r y s t a l l i n i t y (where x c > M.0 ^ s ) , p a r t i a l averaging of a n i s o t r o p i c g and A q u a n t i t i e s occurs (Figure 111-16). I t i s c l e a r then, from both Figures 111-15 and 16, that as molecular tumbling i s slowed, the high f i e l d l i n e begins to broaden followed by the low f i e l d l i n e and then the c e n t r a l l i n e . Tumbling of a molecule can be both i s o t r o p i c (tumbling e q u a l l y about a l l axes) or a n i s o t r o p i c ( r e s t r i c t e d motion about some axes), however, c o n t r i b u t i o n s by a n i s o t r o p i c motion w i l l not be discussed here. Figure 111-17 shows a s e r i e s of spectra obtained from a system where the i s o t r o p i c n o t i o n i s decreasing and c o r r e l a t i o n times are i n c r e a s i n g . Again, as the motion i s reduced, the high f i e l d l i n e broadens before the c e n t r a l or low f i e l d l i n e s u n t i l i n ( f ) the spectrum resembles the poly-c r y s t a l l i n e spectrum where the s p l i t t i n g 2A can c l e a r l y be seen. This l i n e broadening e f f e c t , caused by reduced motion, can be a s e n s i t i v e probe f o r the s i z e of the molecule c o n t a i n i n g the n i t r o x i d e . In Figure 111-18 the s o l u t i o n spectrum of compounds [11] and [36] are compared. Here the d i f f e r e n c e between molecular weights of 172 and 976 can e a s i l y be seen i n the broadening and subsequent r e d u c t i o n i n the peak-to-peak height of the high f i e l d l i n e . From such spectra one can a l s o c a l c u l a t e the c o r r e l a t i o n time x f o r the molecule. c The peaks i n the s p e c t r a of i s o t r o p i c a l l y tumbling molecules are assumed to be l o r e n t z i a n . When t h i s i s the case the peak-to-peak height and l i n e width are f u n c t i o n s of the transverse r e l a x a t i o n time T2-28 Stone et a l . have shown t h i s as given by 169 20 G Figure 111-17. Esr spectra of [11] H0S£ i n aqueous g l y c e r o l s o l u t i o n s where the v i s c o s i t y increases and the temperature lowered from ( a ) - ( f ) (adapted from reference (21) Swartz, Bolton & Borg). Figure 111-18. Esr spectrum of d i l u t e methanol s o l u t i o n s of the n i t r o x -ide [11] H0S2. (upper spectrum) and the n i t r o x i d e [36] (lower spectrum). 171 [ T o . ^ ) ] " 1 = x c{ [31(1+1) + 5m^]^+ | ( A Y H 0 ) 2 - j^kyU^} + X (13) where = c o r r e l a t i o n time, I , m = nuclear angular momentum quantum numbers, Hq = a p p l i e d f i e l d strength and b = 4 r ( A - A ) i n H (14) 3 zz xx z A ^ = " l f [ S z z - i ( g x x + g y y ) ] (15) X i s a f u n c t i o n which accounts f o r other c o n t r i b u t i o n s to the l i n e width. This expression i s only v a l i d under the f o l l o w i n g c o n d i t i o n s : (1) the h y p e r f i n e i n t e r a c t i o n i s a x i a l l y symmetric, i . e . , A^ x = A y y J (2) the 2 2 molecular motion i s i s o t r o p i c and s u f f i c i e n t l y slow that co >> 1 (where ui = 8eH 0/h and b 2 T 2 << 1 ) , so that l i n e widths are i n f l u e n c e d ; , 2 and (3) that i << 1, which ensures that the three l i n e s do not over-l a p . Thus at X band the equations are a p p l i c a b l e i n the range -11 -9 5 x 10 < < 5 x 10 s. U s u a l l y f o r n i t r o x i d e s i n low v i s c o s i t y s o l v e n t s these c o n d i t i o n s are met so that i f we set 1 = 1 f o r n i t r o g e n i n (13) the f o l l o w i n g expression i s obtained: T 2(0) , b 2 2 « 1 " x c T 2 ( 0 ) [ I5bA Y H 0m I - g m^ (16) The r a t i o T2(0)/T2(m ) can be expressed i n terms of the r a t i o of the peak-to-peak heights by T 2(0) fx-- ^ (17) where h i s the peak-to-peak height i n a r b i t r a r y u n i t s . T 2(0) i s m I r e l a t e d to the l i n e widths by T 2 ( 0 > = 7?3lbw ( 1 8 ) where Av(0) i s the l i n e width f o r the c e n t r a l peak i n H2- I f we then 172 i n s e r t (17) and (18) i n t o (16) we get ho t 2 — = 1 [Cjm + C2m ] (19) m TT/3AV(0) where Ci = •—-bAyHo (20) C 2 = b 2/8 (21) I t i s now p o s s i b l e to solve f o r x w i t h m_ = +1 and mT = -1 i n terms of v c I I or C 2. Values of x c derived w i t h C\ have been found to be very 21 dependent on the microwave power so that i t i s best to avoid using Cj l e s s the microwave dependence i s known. Therefore we solve (19) f o r un x i n terms of Co c c hi -± 2 h ° J- h0 i 9/3A\)C0i h l h - l 4^(A -A ) 2 ZZ XX Because i t i s e a s i e r to measure the l i n e widths i n gauss than i n h e r t z we convert Av(0) to AH(0) and A - A from h e r t z to gauss by Eq. (1) w i t h g taken as the averaged g value. In gauss Eq. (23) i s : _ r r h (\i . , V>i ? 1 9/3h AH(0) , . T c " [(h7} + ^ " 2 1 4^gB (A -A ) 2 ( 2 4 )  1 -1 & e zz xx 27 Then using A =31.1 gauss, A = 5 gauss, andg=2.0059 i n Eq. (24) Z Z XX values of x c f o r compound [11] and [36] i n methanol are c a l c u l a t e d to be 1.4 x 1 0 1 1 s and 1.1 x 1 0 1 0 s r e s p e c t i v e l y . The assumption of i s o t r o p i c tumbling f o r compound [36] may be u n r e a l i s t i c but these f i g u r e s , however, seem i n t u i t i v e l y reasonable. Under the author's guidance, a f o u r t h year student, A r t van der E s t , c a l c u l a t e d the TQ values (Table I I I - 4 ) f o r the f o l l o w i n g compounds i n chloroform. Again, these numbers f o l l o w the 173 TABLE I I I - 4 : C o r r e l a t i o n times f o r n i t r o x i d e s i n chloroform s o l u t i o n Compound ^ ( s ) MW 10 2.38 x 10 1 1 171 12 39 6.46 x 10 1 1 319 9.44 x 1 0 - 1 1 711 expected order and approximate magnitudes. The value f o r s y n t h e t i c g l y c o l i p i d compound [39] a l s o provides some evidence f o r the proof of s t r u c t u r e s i n c e x c c l e a r l y i n d i c a t e s [39] i s a much l a r g e r molecule than i t s precursor [12]. As w e l l as l i n e broadening due to A and g a n i s o t r o p i e s there can a l s o be broadening due to e i t h e r d i p o l a r or exchange, e l e c t r o n - e l e c t r o n i n t e r a c t i o n s . Both are dependent upon the conc e n t r a t i o n of m o b i l i t y of the n i t r o x i d e . D i p o l a r broadening i s due to the d i p o l e - d i p o l e i n t e r a c -t i o n between e l e c t r o n s p i n s . Each unpaired e l e c t r o n " f e e l s " a l o c a l i z e d f i e l d due to the motions of a l l of the other e l e c t r o n s . In a randomly 174 o r i e n t e d sample, where the molecular motion i s r a p i d , the d i p o l a r e f f e c t s are average to zero; however, i n an o r i e n t e d sample where the motion i s slow, each e l e c t r o n experiences a s l i g h t l y d i f f e r e n t f i e l d due to i t s l o c a l environment. C l e a r l y the magnitude of t h i s e f f e c t i s dependent ' upon the separation between adjacent unpaired e l e c t r o n s and hence depends on the c o n c e n t r a t i o n . Thus i f we can measure the d i p o l a r broadening we can c a l c u l a t e the d i s t a n c e between adjacent r a d i c a l s . D i p o l a r broaden-in g i s a l s o temperature dependent and as the temperature r i s e s the d i p o l a r e f f e c t s begin to average. The other form of e l e c t r o n - e l e c t r o n i n t e r a c t i o n , that can a f f e c t l i n e widths, i s e l e c t r o n exchange. This e f f e c t can be i l l u s t r a t e d c l e a r l y i n Figure 111-19. For s i m p l i c i t y l e t us consider the r a d i c a l to e x i s t i n two s t a t e s , A and B, such that the g f a c t o r i s d i f f e r e n t i n the two s t a t e s and the s e p a r a t i o n between the two resonant f i e l d s f o r A and B i s 6H. As the e l e c t r o n s begin to exchange between the two s t a t e s A and B, that i s , A±^B the s p e c t r a i n (b)-(e) w i l l r e s u l t . I f the exchange rat e R i s f a s t e r than the frequency d i f f e r e n c e between A and B, that i s where R < —^g-, then we ^e w i l l see an average of the two s i g n a l s as i n (d) and (e) and the l i n e i s s a i d to be exchange narrowed. I f , on the other hand, the r a t e i s much . slower than t h i s s p l i t t i n g , that i s where R > ——-, then the spectrum i n (b) i s obtained and the l i n e s are s a i d to be exchange broadened. When R ^ —^TJ we can no longer d i s t i n g u i s h between i n d i v i d u a l s t a t e s and the ' e spectrum i n (c) r e s u l t s . This model system can a l s o be used to e x p l a i n the e f f e c t s of the tumbling r a t e T c on the l i n e widths of the n i t r o x i d e s p e c t r a , i . e . , averaging or non averaging of g and A a n i s o t r o p i e s . 175 Figure 111-19. E f f e c t s of the r a t e of exchange between A and B on the esr spectra (a) no exchange, (b) slow exchange, (c) intermediate exchange,' (d) and (e) f a s t exchange (exchange narrowing) (adapted from reference (21) Swartz, Bolton & Borg). 176 From the d i p o l a r i n t e r a c t i o n the d i s t a n c e between n i t r o x i d e s can be measured, as was shown p r e v i o u s l y i n s e c t i o n I I I C ( i ) ( d ) . I f we assume a random three dimentional d i s t r i b u t i o n of spins we can r e l a t e the mean nearest neighbour distance to the d e n s i t y of s p i n l a b e l s by: r = ( ^ p ) " 1 / 3 r ( | - ) (24) where p i s the d e n s i t y i n nm 3 , r i s the gamma f u n c t i o n r(n) = / q e X x n "'"dx and r e | ) = 0 . 8 9 2 6 1 2 9 , thus r = 0 . 5 5 3 7 3 p ~ 1 / 3 (25) The d e n s i t y i s r e l a t e d to the molar con c e n t r a t i o n by: p = 0 .602 [SZ] (26) where [SH] i s the molar concentration of s p i n l a b e l . S u b s t i t u t i n g Eq. (26) i n t o (25) we get: r = 0 .656 [ S £ ] " 1 / 3 Measuring the e f f e c t i v e c oncentration of s p i n l a b e l s i n the l a b e l l e d sample cannot be done d i r e c t l y , however, s i n c e the d i p o l a r broadening i s concentration dependent i t can be measured i n d i r e c t l y . The s p e c t r a l parameter which i s a measure of the broadening i s d^/d, where dj i s the t o t a l i n t e n s i t y of the extreme components of the spectrum at 77 K, and d i s the i n t e n s i t y of the c e n t r a l peak as shown i n Figure I I I - 5 . Using homogeneous s o l u t i o n s of v a r y i n g concentrations f o r both 2 , 2 , 6 , 6 -t e t r a m e t h y l p i p e r i d i n e - l - o x y l i n methanol and 4-amino - 2 , 2 , 6 , 6-tetramethyl-p i p e r i d i n e - l - o x y l i n aqueous g l y c e r o l , Drs. J . D. A p l i n and J . C. Waterton found that at 77 K, d^/d i s r e l a t e d to the c o n c e n t r a t i o n by: dl d! d d <»dil Figure I I I - 5 . Powder spectrum showing the heights dj and d and the s p l i t t i n g 178 I t was found that when the concentration i s reduced below a c r i t i c a l v a l u e ' v 5 x 10 M no d i p o l a r broadening e x i s t s and dj/d remains constant This i s the value of d i / d at i n f i n i t e d i l u t i o n (d-i/d) ,... . This number v a r i e s somewhat from system to system and i s a f u n c t i o n of solvent p o l a r i t y and the amount of r e s i d u a l motion at 77 K. T y p i c a l values are c l o s e to 0.4. Combining Eq. (27) and (26) g i v e s : d l d l _ _3 T = ( T ) ... +0.58 r J (28) d d °°dil This equation can then be used f o r any system to c a l c u l a t e d i s t a n c e s between n i t r o x i d e s . The d i s t a n c e between n i t r o x i d e s i n the b i r a d i c a l (25) can a l s o be c a l c u l a t e d using t h i s technique. The distances between n i t r o x i d e s of a N ^ N CI [25] 23 v a r i e t y of b i r a d i c a l s have p r e v i o u s l y been determined by Kokorin et a l . 30 and K a l i k o v et a l . u s i n g , i n p r i n c i p a l , the same e m p i r i c a l method. In the distance measurement of (25) a (d-i/d) value was obtained rodil by u s i n g the dx/d value (0.467) f o r C£2T0S£ [22] at 2 x 1 0 _ 4 M i n c h l o r o -form at 77 K. The b i r a d i c a l must a l s o be run at i n f i n i t e d i l u t i o n as w e l l so that only d i p o l a r i n t e r a c t i o n s between n i t r o x i d e s w i t h i n the same molecule are detected. A value of 0.813 f o r the d^/d of (25) i n a -4 2 x 10 M chloroform s o l i d s o l u t i o n at 77 K, gave a b i r a d i c a l distance 179 of 1.19 nm. The f r o z e n s p e c t r a of the monoradical [22] and the b i r a d i c a l {253 are shown i n Figure 111-20. The room temperature s o l u t i o n spectrum of b i r a d i c a l s can a l s o be very i n t e r e s t i n g . The s o l u t i o n spectrum of [25] shows (Figure 111-21) f i v e l i n e s as opposed to three f o r a monoradical, because there are two n i t r o g e n s p i n 1 atoms present and the t o t a l s p i n quantum number takes a l l i n t e g e r values from 2 to -2. The f i v e l i n e s are only seen, however, i n b i r a d i c a l s that have a high enough frequency of e l e c t r o n exchange between n i t r o x i d e s . I f the frequency of exchange i s greater than the hyperfine i n t e r a c t i o n frequency, the mean residence time of each e l e c t r o n at the two n u c l e i w i l l be the same and the esr spectrum w i l l c o n s i s t of f i v e e q u i d i s t a n t l i n e s w i t h a r a t i o of i n t e n s i t i e s of 1:2:3:2:1. This exchange r a t e i s of course, dependent upon the d i s t a n c e between r a d i c a l s and, as i n the case of compound [25], they are too f a r apart f o r complete exchange and as a r e s u l t the +1 and -1 l i n e s have a very low i n t e n s i t y . 17 27 B i r a d i c a l s a l s o have been used as s p i n probes ' 180 Figure 111-20. 77K i n f i n i t e d i l u t i o n chloroform esr spectra of (A) the monoradical [22] and (B) the b i r a d i c a l [25]. 181 Figure 111-21. Ambient temperature chloroform esr spectra of (A) the monoradical [11] and (B) the b i r a d i c a l [25]. 182 References 1. E. M. Smolin and L. Rapoport, The Chemistry of H e t e r o c y c l i c Com-pounds, 1_3, 1959. 2. A. F u r s t , Chemistry of C h e l a t i o n i n Cancer, Charles C. Thomas (pub-l i s h e r ) , S p r i n g f i e l d , I l l i n o i s , 1963. 3. W. R. S t i n e , Chemistry f o r the Consumer, A l l y n and Bacon, Inc., Toronto, 1978. 4. R. L. M. A l l e n , Color Chemistry, Appelton-Century-Crofts, New York, 1971. 5. H. J . Bohme, G. Koppershlager, J . Schulz and E. Hofmann, J . Chroma-t o g r . , 69, 209(1972). 6. T. Linnas, Tezisy Dokl.-Resp. Konf. Molodykh Uch.-Khim, 2nd, 1_, 137(1977), CA 89, 15-125274M; V. Fedoseev, M. Pank, H. H e i n l o , A. Murel, 0. K i r r e t , E e s t i Nsv. Tead. Akad. Toim., Keem, Geol., 26, 326(1977), CA 88, 05-33855C. 7. A. A. Malinauskas and J . J . Kulyus, B i o t e c h n o l . Bioeng., 20, 769(1978), CA 8901-2328Z. 8. J . Danner, H. M. Lenhoff, W. Heagy, J . Solid-Phase Biochem. , 1_, 177(1976), CA 8821-148369Q. 9. D. B l a k e s l e e , J . Immunol. Methods, 17, 361(1977). 10. A. Abuchowski, T. Van Es, N. C. Paczuk and F. F. Davis, J . B i o -l o g i c a l Chem., 252, 3578(1977); A. Abuchowski, J . R. McCoy, N. C. Palczuk, T. van Es and F. F. Davis, i b i d . , 252, 3582(1977). 11. M. S. Wrighton, M. C. P a l a z z o t t o , A. B. B o c a r s l y , J . M. B o l t s , A. B. F i s h e r and L. Nadjo, J . Am. Chem. S o c , 100, 7264(1978); J . M. B o l t s , A. B. B o c a r s l y , M. C. P a l a z z o t t o , E. G. Walton, N. S. Lewis and M. S. Wrighton, i b i d , 101, 1378(1979); Chem. Eng. News, Mar. 19, 1979, p. 25; A. W. C. L i n , P. Yeh, A. M. Yacynych and T. Kuwana, J . E l e c t r o a n a l . Chem., 84, 411(1977). 12. R. J . F i e l d e r , C. T. Bishop, S. F. Grappel and F. Blank, J . Immun-ology, 105, 265(1970). 13. C. T. Bishop and A. S. Chaudhari, Can. J . Chem., 50, 1987(1972); R. R. King, F. P. Cooper and C. T. Bishop, Carbo. Res., 55, 83(1977). 14. R. T. Morrison and R. N. Boyd, Organic Chemistry, T h i r d E d i t i o n , A l l y n and Bacon, New York, 1973. 15. G. I. L i k h t e n s h t e i n and P. K. H. Bobodzhanov, B i o f i z i k a , 14, 783(1969). 183 16. Arthur van der E s t , B.Sc. t h e s i s , U n i v e r s i t y of B r i t i s h Columbia, 1979. 17. V. N. Parmon, A. I . Kokorin and G. M. Zhidomirov, Zhurnal Struk-t u r n o l K h i m i i , 18, 132 (104 t r a n s l a t e d ) (1977). 18. R. M. Marupov, P. K. H. Bobodzhanov, N. V. Ko s t i n a and A. B. Shapiro, B i o f i z i k a , 21, 825 (848 t r a n s l a t e d ) (1976). 19. E. G. Rosantzev, Free N i t r o x y l R a d i c a l s , H. U l r i c h ( t r a n s l a t o r and Ed.), Plenum, London, 1970; B. J . Gaffney, i n L. J . B e r l i n e r , J r . (Ed.), Spin L a b e l l i n g , Theory and A p p l i c a t i o n s , Academic Press, New York, 1976. 20. J . D. A p l i n and L. D. H a l l , J . Am. Chem. S o c , 99^ , 4162(1977); Carbohydrate Research, 59, C20(1977); J . Am. Chem. S o c , 100, 1934(1978). 21. J . E. Wertz and J . R. B o l t o n , E l e c t r o n Spin Resonance, Elementary Theory and P r a c t i c a l A p p l i c a t i o n s , McGraw-Hill, New York, 1972; J . R. B o l t o n , D. Borg and H. Swartz, B i o l o g i c a l A p p l i c a t i o n s of  E l e c t r o n Spin Resonance, W i l e y - I n t e r s c i e n c e , 1972. 22. J . D. A p l i n and L. D. H a l l , J . Am. Chem. Soc. ( i n p r e s s ) ; J . C. Waterton and L. D. H a l l , J . Am. Chem. S o c , 101, 3697(1979). 23. A. L. Kokorin, K. I . Zamarayer, G. L. Grigoryan, V. P. Ivanov and E. G. Rosantzev, B i o f i z i k a , 17, 34 (39 i n t r a n s l . ) (1972). 24. D. A. Rees, Polysaccharide Shapes, Chapman and H a l l , London, 1977; R. L. w h i s t l e r , Ed., Methods i n Carbohydrate Chemistry, V o l . I l l , Academic Press, New York, 1963. 25. J . F. W. Keana i n Spin I I L a b e l l i n g , Theory and A p p l i c a t i o n s , L. J . B e r l i n e r (Ed.), Academic Press, New York, 1979. 26. J . D. A p l i n , Ph.D. t h e s i s , U n i v e r s i t y of B r i t i s h Columbia, 1979. 27. L. J . B e r l i n e r (Ed.), Spin L a b e l l i n g , Theory and A p p l i c a t i o n s , Academic Press, New York; 1976. 28. T. J . Stone, T. Buckman, P. L. Nardio and H. M. McConnell, Proc. N a t l . Acad. S c i . USA, 54, 1010(1965). 29. R. C. Weast (Ed.), CRC Handbook of Chemistry and P h y s i c s , CRC Pr e s s , Cleveland, Ohio, 58th E d i t i o n , 1977. 30. A. V. K u l i k o v , G. I . L i k h t e n s h t e i n , E. G. Rozantzev, V. I. Suskina and A. B. Shapiro, B i o f i z i k a , 1J7_, 42 (40 i n t r a n s l . ) (1972). CHAPTER IV SUMMARY A b i l i t y to chemically modify carbohydrates, i n a v a r i e t y of d i f f e r -ent ways, i s of great importance and i n t e r e s t . In the context of the present study the term "chemical m o d i f i c a t i o n " r e f e r s mainly to the a t t a c h -ment of chemical l i g a n d s which possess unique s p e c i f i c p r o p e r t i e s r a t h e r than i n the sense of blocking-group technology or other p u r e l y s y n t h e t i c carbohydrate procedures. I t was the i n t e n t i o n of t h i s work to syn t h e s i z e m a t e r i a l s which possess the unique c h a r a c t e r i s t i c s of carbohydrates, plus the eq u a l l y unique p r o p e r t i e s of the appended group of i n t e r e s t . I t i s hoped that such m a t e r i a l s and the un d e r l y i n g chemical technology f o r t h e i r production w i l l have broad a p p l i c a t i o n s i n a v a r i e t y of areas of chemistry, some of which have been a l l u d e d to i n the te x t of t h i s t h e s i s . In chapter I the s y n t h e s i s of metal c h e l a t e complexes of sugars was demonstrated by the formation of S c h i f f ' s base complexes derived from amino sugars and e i t h e r s a l i c y l a l d e h y d e or 3-formyl-2-hydroxy-benzoic a c i d . A v a r i e t y of novel complexes were formed, mostly i n v o l v i n g copper ( I I ) , i n c l u d i n g a b i n u c l e a r complex and a w a t e r - s o l u b l e , sugar-copper complex. The main impetus behind the work i n chapter I , and a l s o that i n chapter I I , was the hope that the combined presence of sugar and metal moieties would impart to these compounds i n t e r e s t i n g b i o l o g i c a l p r o p e r t i e s , and hence that t h i s chemistry may be u s e f u l i n the formation of pharmaceuticals. U n f o r t u n a t e l y , time d i d not permit f o r the 184 185 e v a l u a t i o n of those p r o p e r t i e s , or indeed of the other numerous, poten-t i a l a p p l i c a t i o n s of t h i s chemistry o u t l i n e d i n the i n t r o d u c t i o n s to these chapters. The work i n chapter I I represents an e x t e n t i o n of chapter I to i n c l u d e organometallic ir-complexes. S p e c i f i c a l l y , ferrocene mono-saccharide complexes were synthesized to y i e l d both organic- and water-s o l u b l e compounds. Here too, i t i s not naive to suggest numerous a p p l i c a t i o n s i n the pharmaceutical i n d u s t r y where ferrocene i s i d e a l because of i t s low t o x i c i t y and the ease of s u b s t i t u t i o n of t h i s mole-cul e . Thus, ferrocene d e r i v a t i v e s of p e n i c i l l i n and cephalosporin have been prepared; some e x h i b i t high a n t i b i o t i c a c t i v i t y w h i l e others have proven to be potent B-lactamase inhibitors"'". Some ferrocene d e r i v a t i v e s 2 are a l s o known to be u s e f u l as hematinic agents. Again, as suggested i n the i n t r o d u c t i o n to that chapter, there are a l s o numerous other pos-s i b l e a p p l i c a t i o n s of t h i s chemistry. I t i s a l s o important to note that i t was found that proton s p i n l a t t i c e r e l a x a t i o n r a t e s could be used to as s i g n the proton resonances of the s u b s t i t u t e d c y c l o p e n t a d i e n y l r i n g of ferrocene d e r i v a t i v e s . This technique, i s of course, not r e s t r i c t e d to sugar f e r r o c e n y l compounds but can be a p p l i e d to any ferrocene d e r i v a t i v e . Indeed i t should f i n d widespread use i n numerous other f a m i l i e s of organometallic substances. In chapter I I I , some of the p o t e n t i a l l i m i t a t i o n s of the chemistry described i n chapters I and I I were explored by the i n v e s t i g a t i o n of the re a c t i o n s of cyanuric c h l o r i d e w i t h carbohydrates. With t h i s reagent i t was not only p o s s i b l e to couple metals to monosaccharides, but a l s o to l i n k a v a r i e t y of other substances, i n c l u d i n g s p i n l a b e l s and hydro-phobic a l k y l groups to both mono- and poly-saccharide f a m i l i e s of carbo-186 hydrates. The main t h r u s t of t h i s chapter i n v o l v e d the n i t r o x i d e s p i n l a b e l l i n g of these carbohydrates v i a cyanuric c h l o r i d e . This was shown to be important to f u t u r e work i n t h i s area since the s p i n l a b e l group can r e l a y a great d e a l of informat i o n about the i n t e r a c t i o n of cyanuric c h l o r i d e and i t s d e r i v a t i v e s w i t h p o l y s a c c h a r i d e s . I t i s often p a r t i c u l a r l y d i f f i c u l t to c h a r a c t e r i z e chemical r e a c t i o n s of macro-molecular d e r i v a t i v e s , e s p e c i a l l y when the l e v e l of d e r i v a t i z a t i o n i s low, and knowledge gained from s p i n l a b e l l i n g can be very h e l p f u l i n t h i s respect. Although f e r r o c e n e - t r i a z i n e d e r i v a t i v e s and a chromium organometallic t r i a z i n e d e r i v a t i v e were synthesized i n chapter I I , no d i r e c t l i n k between t h i s chapter and the work i n chapter I was made. However, the combination of these two areas, whereby metal chelate l i g a n d s can be attached to the t r i a z i n e r i n g , l e a v i n g at l e a s t one d i s p l a c e a b l e c h l o r i n e s u b s t i t u e n t , could w e l l lead to some very u s e f u l compounds. Metal chelate reagents produced from t h i s combination should f i n d many a p p l i c a t i o n s i n areas such as a f f i n i t y chromatography and polymer supported heterogeneous c a t a l y s i s to name j u s t two. As was a l s o demonstrated, t h i s s_-triazine chemistry i s not l i m i t e d to carbo-hydrates but can be extended to the d e r i v a t i z a t i o n of other m a t e r i a l s such as p r o t e i n s , alumina and g l a s s . Many extentions of the present s t u d i e s whether i n whole, or i n p a r t , may t h e r e f o r e be expected both i n the carbohydrate f i e l d and i n other areas. In r e t r o s p e c t , i t i s a p i t y that time d i d not permit the author to have eva l u a t i o n s made of the p r o p e r t i e s of some of the substances synthesized here. Those evalua-t i o n s , together w i t h the extensions a l l u d e d to above could w e l l , however, c o n s t i t u t e the research work of a considerable army of graduate students; c e r t a i n l y t h e i r e v a l u a t i o n f a r exceeds the scope of one student, even i n t h i s l a b o r a t o r y ! 187 References 1. E. I . Edward, R. Epton and G. Marr, J . Organomet. Chem., 167, 53 (1979). 2. J . C. Johnson, Metallocene Technology, Noyes Data Corporation, Park Ridge, New Jersey, 1973. CHAPTER V EXPERIMENTAL VA: E l e c t r o n Spin Resonance Esr s p e c t r a were recorded at X-band using a V a r i a n E-3 instrument i n the d e r i v a t i v e a bsorption mode and i n t e g r a t e d using a P a c i f i c P r e c i -s i o n Co. MO-1012A i n t e g r a t o r . The second i n t e g r a t i o n was performed by peak c u t t i n g and weighing and comparison w i t h f r e s h l y - p r e p a r e d standard s o l u t i o n s of the 4-hydroxy n i t r o x i d e [11]. Spectrometer s e t t i n g s — modulation amplitude, f i l t e r time constant and scan r a t e — w e r e chosen i n each case to avoid d i s t o r t i o n of the s p e c t r a l l i n e s , and power l e v e l s were non s a t u r a t i n g . The ambient temperature was always 25°C + 1, and the f i e l d always increased from l e f t to r i g h t . L ine widths were measured using the s m a l l e s t p o s s i b l e scan range ( g e n e r a l l y not that shown i n the diagrams) and at l e a s t two measurements were made using d i f f e r e n t scans i n each case. The f i e l d was c a l i b r a t e d using a proton nmr magnetometer and the X-band microwave frequency was monitored on a Hewlett-Packard 5245-L e l e c t r o n i c counter equipped w i t h a 8-18 GHz frequency converter. The resonance frequency i s dependent upon the a p p l i e d f i e l d : most experiments, i n c l u d i n g the ones described h e r e i n , were conducted at X-band (about 9.1-9.5 HGz), which corresponds to an e x t e r n a l f i e l d of the order of 3-3.4 KG. In the experiments described here, net absorption of micro-wave energy from Hi occurs at resonance as a r e s u l t of the great propor-t i o n of spins present i n the lower energy s t a t e . Absorption of energy i s 188 189 monitored w i t h the a i d of phase s e n s i t i v e d e t e c t i o n using f i e l d modulation at 10 5 Hz (provided by Helmholz c o i l s on each s i d e of the c a v i t y and recorded as the f i r s t d e r i v a t i v e of the absorption s i g n a l ) . Spectra at 77K were obtained using a Dewar i n s e r t c o n t a i n i n g l i q u i d n i t r o g e n , at 0.16 mW microwave power, the lowest a v a i l a b l e . Oxygen was prevented from condensing i n the sample tube by s e a l i n g the top w i t h a rubber septum cap. A l l low temperature sp e c t r a were recorded i n 3 mm i . d . quartz tubes when non p o l a r organic solvents were used and i n 1 mm i . d . Pyrex tubes f o r aqueous s o l u t i o n s . At room temperature, both these types of tubes were used along w i t h a f l a t h i g h - q u a l i t y quartz c e l l , c a p a c i t y 73 uL, w i t h ground glass j o i n t s at both ends ( J . Scanlon Co.). The 3 mm i . d . tube was p r e f e r r e d f o r use w i t h the d i l u t e chloroform s o l u -t i o n s of the copper complexes s i n c e more sample could be placed i n the esr c a v i t y thereby g i v i n g a b e t t e r s i g n a l to noise r a t i o . Powder samples of metal complexes were run i n s i d e m e l t i n g p o i n t c a p i l l a r y tubes placed i n s i d e a 3 mm i . d . quartz tube. A t e f l o n i n s e r t designed by Dr. F. G. H e r r i n g was used f o r aqueous s l u r r i e s of water i n s o l u b l e p o l y -saccharides. This c o n s i s t e d of a c y l i n d e r of diameter 10 mm w i t h a h a l f -c y l i n d e r s e c t i o n 30 mm long cut away i n the center, forming a f l a t surface 10 x 30 mm upon which the wet polysaccharide was placed beneath a g l a s s cover s l i p , the l a t t e r r e t a i n e d by surface t e n s i o n . S o l u t i o n s of n i t r o x i d e s , whose spec t r a were to be used f o r c o r r e l a t i o n time ( T ) measurements, were deoxygenated by bubbling n i t r o g e n through f o r s e v e r a l minutes. 190 VB: Nmr Measurements Proton nmr spec t r a were measured at 270 MHz w i t h a protype of a home-built spectrometer based on a Bruker WP-60 console, a N i c o l e t 1180 computer (32K), a N i c o l e t 293A pulse c o n t r o l l e r u n i t , a Dia b l o Disk, and an Oxford Instruments Superconducting s o l e n o i d . Concentrations of normal samples ranged from 0.01 to 0.10 M and measurements were performed i n concentrations of < 0.5 M, w i t h degassing achieved by f i v e f r e e z e pump thaw c y c l e s . A l l deuterated solvents were obtained from Merck Sharp and Dohme (Montreal, Canada) and t e t r a m e t h y l s i l a n e was used as a standard. R e l a x a t i o n data were obtained using the standard N i c o l e t software f o r the phase a l t e r n a t i n g i n v e r s i o n recovery experiment (180°-t-90°-Acq-Delay-180° -t-90°-Acq-Delay) , and the R e v a l u e s were X x *~x x n/^-c a l c u l a t e d using a X-nCM^ -M^ ) vs t plot''". Only p r e - n u l l point r e l a x a t i o n data were used i n these p l o t s to accommodate the i n i t i a l slope approxima-. 2 t i o n . VC: General Synthetic Procedures A l l s o l u t i o n s were concentrated using a Buchi r o t a r y evaporator. A l l m e l t i n g p o i n t s were determined us i n g a Thomas Hoover Unimelt i n s t r u -ment (Model 6406-K) and are c o r r e c t e d . A l l o p t i c a l r o t a t i o n s were determined using a Perkin-Elmer P o l a r i m e t e r (model 241-MC). Thin l a y e r chomatography ( t i c ) was performed on s i l i c a g e l p l a t e s ( B a k e r - f l e x S i l i c a g e l 1B2-F) using the f o l l o w i n g s o l v e n t s : (A) 1:1, toluene: e t h y l acetate; (B) 4:1 toluene: e t h y l a c e t a t e ; (C) 1:5 methanol: chloroform. A l l compounds were checked f o r p u r i t y by t i c using one of these solvent systems. Column chromatography was performed 191 using 100-200 ASTM mesh s i l i c a ( F i s c h e r ) packed i n columns approximately 2.5 x 50 cm, and e l u t e d w i t h s o l v e n t s (A), (B), or (C). For r e a c t i o n s r e q u i r i n g anhydrous (dry) s o l v e n t s the solvents were d r i e d by standard methods, d i s t i l l e d and stored under a n i t r o g e n atmosphere. The source of a l l chemicals and m a t e r i a l s w i l l be given i n the experimental s e c t i o n of each chapter. A l l m i c r o a n a l y s i s was performed by Mr. P. Borda, of t h i s department. The mass s p e c t r a were obtained using an A t l a s CH-4B mass spectrometer and high r e s o l u t i o n determinations were obtained using an AE1 MS-9 or an MS-50 mass spectrometer. VD: Chapter I ( i ) Sources of M a t e r i a l s Sources f o r the key chemicals used i n the synth e s i s of compounds i n t h i s chapter are as f o l l o w s : s a l i c y l a l d e h y d e was obtained from F i s h e r and was vacuum d i s t i l l e d before use; s a l i c y l i c a c i d ( M a l i n c k r o d t ) ; hexam-ethylenetetramine (Matheson Coleman and B e l l ) ; cyclohexylamine, t-butylamine, n-butylamine and t-propylamine (Eastman); D-glucosamine hy d r o c h l o r i d e (Sigma); carbobenzyloxy-chloride (CBZ c h l o r i d e ) ( A l d r i c h ) ; anisaldehyde (Matheson Coleman and B e l l ) ; c u p r i c and cobaltous acetate ( F i s h e r ) ; z i n c acetate (Matheson Coleman and B e l l ) ; n i c k e l acetate (Baker). p-acetamido-benzensulphonyl c h l o r i d e was prepared by the method i n Vogel^. ( i i ) L i t e r a t u r e Preparations A b r i e f summary w i l l now be given f o r the sy n t h e s i s of compounds that were repeated from the l i t e r a t u r e . Any problems encountered or changes made i n the r e c i p e s w i l l be noted. 192 The compound, 3-formyl-2-hydroxy-benzoic a c i d [ 2 ] , was prepared by 4 the method of Duff and B i l l s . A t y p i c a l p r e p a r a t i o n i n v o l v e d b o i l i n g s a l i c y l i c a c i d (40 g) w i t h hexamethylenediamine (27 g) i n water. The mixture i s then a c i d i f i e d and the y e l l o w p r e c i p i t a t e e x t r a c t e d w i t h benzene. This e x t r a c t i o n procedure separates the r e s u l t i n g 3-formyl-and 5-formyl-2-hydroxy-benzoic a c i d mixture s i n c e the l a t t e r i s i n s o l u b l e i n benzene. The barium s a l t of the t i t l e compound i s then formed i n b a s i c aqueous s o l u t i o n w i t h subsequent h y d r o l y s i s by HC£ to give the compound i n about a (3 g) y i e l d . The g l y c o s i d e methyl 3,4,6-tri-0-acetyl-2-amino-2-deoxy - 8-D-gluco-pyranoside hydro-bromide [7] was prepared by a combination of r e c i p e s from Chargaff^, Gross^, and I r v i n e and E a r l ^ . F i r s t , the carbobenzyloxy d e r i v a t i v e of glucosamine was prepared by the method of Chargaff^. This product was prepared r e a d i l y by mixing one equivalent of glucosamine h y d r o c h l o r i d e and carbobenzyloxy c h l o r i d e (CBZ c h l o r i d e ) w i t h two equiv-a l e n t s of sodium bicarbonate i n water. The product p r e c i p i t a t e d i n a high y i e l d ^ 90% and was f i l t e r e d and d r i e d . Next the g l y c o s y l bromide was prepared by the method of Gross et a l . . A mixture of a c e t i c a c i d , a c e t i c anhydride, and HBr was added at 0°C to the CBZ glucosamine i n p o r t i o n s and a f t e r 30 minutes HBr gas was bubbled through the mixture r a p i d l y u n t i l the temperature was r a i s e d about 10°C. I f anhydrous c o n d i t i o n s were met the g l y c o s y l bromide u s u a l l y p r e c i p i t a t e d during or s h o r t l y a f t e r the HBr gas was introduced. Ether was then added and a f t e r r e f r i g e r a t i o n f o r a few hours, the product was f i l t e r e d . The g l y c o s y l bromide was r e c r y s t a l l i z e d by d i s s o l v i n g i t i n chloroform at ambient temperature, evaporating t h i s to a small volume w i t h a stream of n i t r o g e n and then adding a small amount of anhydrous ether. The methyl 193 g l y c o s i d e was then formed by the method of I r v i n e and E a r l ^ whereby a 5% s o l u t i o n of the g l y c o s y l bromide i n anhydrous methanol c o n t a i n i n g 1% of anhydrous p y r i d i n e , was l e t stand overnight. The mixture was then evaporated under anhydrous c o n d i t i o n s to a small volume and ether added. Upon r e f r i g e r a t i o n the methyl g l y c o s i d e [7] was obtained. The tetra-O-acetyl-glucosamine d e r i v a t i v e [9] was prepared by the method of Bergman , using anisaldehyde (p-methoxy-benzaldehyde) to pro-t e c t the amino f u n c t i o n . F i r s t , the S c h i f f ' s base was formed by mixing glucosamine h y d r o c h l o r i d e [5] and anisaldehyde i n water w i t h one equiv-a l e n t of sodium bicarbonate. The compound formed r e a d i l y and was f i l t e r e d from the r e a c t i o n mixture. This m a t e r i a l , when dry, was then a c e t y l a t e d i n a 1:1 mixture of a c e t i c anhydride and p y r i d i n e to y i e l d the t e t r a - a c e t a t e [13]. The S c h i f f ' s base was then cleaved by t r e a t i n g the acetate w i t h a d i l u t e aqueous acetone s o l u t i o n of HC£. The s t a r t i n g acetate sugar [13] and s a l i c y l a l d e h y d e were then e x t r a c t e d w i t h c h l o r o -form and the aqueous l a y e r n e u t r a l i z e d w i t h sodium bicarbonate. Once n e u t r a l , the aqueous l a y e r was e x t r a c t e d again w i t h chloroform to give the " f r e e " amino sugar [9]. The product was then r e c r y s t a l l i z e d from an ethanol-water mixture; m.p. 138-139°C. The S c h i f f ' s base s t a r t i n g mate-r i a l that was not hydrolyzed i n i t i a l l y , was recovered from the f i r s t chloroform e x t r a c t i o n and t r e a t e d again w i t h a c i d . The sugar s a l i c y l a l d i m i n e s [ 6 ] , [ 8 ] , and [10] were a l l prepared 9 r e a d i l y by the method of I r v i n e and E a r l by mixing v i g o r o u s l y , the amino sugar and s a l i c y l a l d e h y d e together i n water or i n a methanol water mix-t u r e , w i t h one equivalent of sodium bicarbonate added i n the p r e p a r a t i o n of compounds [6] and [ 8 ] . The mixtures were then f i l t e r e d a f t e r about 3 h and the products obtained i n greater than 80% y i e l d s . R e c r y s t a l -l i z a t i o n from e i t h e r methanol or ethanol gave the pure compounds [6], [8], and [10]. The preparation of the s a l i c y l a l d i m i n e metal complexes Cu (cyclohexyl-sal)2, Cu (n-Bu-sal) 2, Cu ( t - B u - s a l ) 2 and Cu ( i - P r - s a l ) 2 were r e a d i l y prepared by the o r i g i n a l method of S c h i f f and repeated from a more recent reference by Holm"^. Both copper (II) and n i c k e l (II) complexes were prepared i n y i e l d s >^  60% by r e f l u x i n g i n ethanol 0.1 mole of the appropriate n i c k e l or copper salicylaldehyde complex with a 10% mole excess of the amine. The products were obtained by cooling the rea c t i o n mixture and f i l t r a t i o n of the p r e c i p i t a t e . Zinc and cobalt complexes can also be prepared by t h i s method. The salicylaldehyde metal complexes were e a s i l y prepared by mixing salicylaldehyde with the appropriate metal acetate i n aqueous alcohol. ( i i i ) Synthesis Preparation of methy1-3,4,6-tri-0-acetyl-2-deoxy-2-(3-carboxyl-salicylaldimino)-8-D-glucopyranoside [11]. To a s o l u t i o n of amino sugar [7] (0.51 g, 1.27 m mol) i n methanol (4 ml) was added a s o l u t i o n of sodium bicarbonate (0.25 g, 3 m mol) i n H 20 (4 ml) and a solution of 3-formyl-2-hydroxy-benzoic acid (0.22 g, 1.28 m mol) i n methanol (4 ml). The mixture was s t i r r e d at room tem-perature for 1 h and then evaporated to dryness and a small amount of ethanol added.. The r e s u l t i n g p r e c i p i t a t e was f i l t e r e d , washed with cold ethanol and dried under vacuum to give the product i n 70% y i e l d . 22 The compound was r e c r y s t a l l i z e d from ethanol; m.p. 251-252°C, [ a l n + 245.0° ( C 2 C H C £ 3 ) . Anal, calcd. f o r C 2 i H 2 5 N i 0 1 1 : C 53.94, H 5.39, N 2.99; found: C .53.52, H 5.54, N 2.80. 195 P r e p a r a t i o n of S c h i f f ' s Base Metal Complexes from Ligands [ 6 ] , [ 8 ] , [10], and [11]. Ligand [6] was d i s s o l v e d i n methanol at room temperature and lig a n d s [8], [10], and [11] were d i s s o l v e d i n hot ethanol. To these s t i r r e d mix-tures was then added an a l c o h o l i c s o l u t i o n of the appropriate metal acetate. The complex then p r e c i p i t a t e d e i t h e r immediately or upon c o o l -i n g and the products were f i l t e r e d and p u r i f i e d as shown below. Bis-(N-methyl-3,4,6-tri-0-acetyl-2-deoxy-B-D-glucopyranosy1-2-s a l i c y l a l d i m i n o ) Cu ( I I ) [16] was obtained i n 90% y i e l d and was a n a l y t i c -a l l y pure d i r e c t l y from the r e a c t i o n mixture. Slow c r y s t a l l i z a t i o n from an acetone s o l u t i o n y i e l d e d dark brown cubic c r y s t a l , m.p. 250-251°C, [ a ] 2 2 + 777° (C 0.74 CHC£ 3). Anal. Calcd. f o r Ci+oH^CuN^! 8: C 52.91, H 5.29, N 3.08; found: C 53.08, H 5.21, N 3.02, ms (low r e s o l u t i o n ) c a l c d . f o r C i + 0 H i + 8 C u N 2 0 1 8 : 907 ( C u 6 3 ) , 909 (C u 6 5 ) amu (M:M+1 :M+2 :M+3; 100:40:50:20); found: 907 and 909 amu (M:M+1:M+2:M+3; 100:40:50:20). Bis-(N-methyl-3,4,6-tri-0-acetyl-2-deoxy -8-D-glucopyranosyl-2-s a l i c y l a l d i m i n o ) (Zn ( I I ) [17] was obtained i n 41% y i e l d from acetone/ chloroform, m.p. 272-273°C, [ a ] 2 2 + 240.3° (C 0.77 CHC£ 3). Anal, c a l c d . f o r C i + 0 H i + 8 N 2 0 1 8 Z n : C 52.80, H 5.28, N 3.08; found: C 53.00, H 5.40, N 3.07; ms (low r e s o l u t i o n ) c a l c d . f o r C i + 0 H i + 8 N 2 0 1 8 Z n : 908 (Zn 6* 4) , 910 ( Z n 6 6 ) , 911 ( Z n 6 7 ) , 912 ( Z n 6 8 ) amu (M:M+1:M+2:M+3:M+4; 100:44:65:33:46); found: 908, 910, 911, 912 amu (M:M+1:M+2:M+3:M+4; 100:50:70:30:45). B i s - (N-methyl-3 ,4,6-tri-0_-acetyl-2-deoxy- 8-D-glucopyranosyl-2-s a l i c y l a l d i m i n o ) Co ( I I ) [18] was obtained i n 58% y i e l d from chloroform, m.p. 260-261°C, [ a ] 2 2 - 25.6° (C 0.39 CHC£ 3). Anal, c a l c d . f o r Ci^H^e CoN 20 1 8: C 53.18, H 5.31, N 3.10; found: C 53.06, H 5.39, N 3.20; ms 196 (low r e s o l u t i o n ) c a l c d . f o r Ci+gHi^Co^Oig: 903 amu; found 903 amu. Bis-(N - l , 3 , 4 , 6 - t e t r a - 0 - a c e t y l - 2 - d e o x y - 8 - p - g l u c o p y r a n o s e - 2 - s a l i c y l -aldimino) Cu ( I I ) [19] was obtained i n 90% y i e l d from ethanol, m.p. 125-126°C, [ a ] 2 2 + 194° (C 0.5 CHC£ 3). Anal, c a l c d . f o r C i t 2 H i t 8 C u N 2 0 2 0 : C 52.30, H 4.98, N 2.90; found: C 51.90, H 5.03, N 2.84; ms (low r e s o l u t i o n ) c a l c d . f o r C ^ H ^ C U N ^ Q = 963 ( C u 6 3 ) , 965 (C u 6 5 ) amu (M:M+1 :M+2 :M+3; 100: 40:50:20); found: 963, 965 amu (M:M+1:M+2:M+3; 100:40:50:20). (N-2-amino-2-deoxy-a,g-D-glucopyranose-salicylaldimino) Cu ( I I ) [21] was prepared i n 67% y i e l d , m.p. 150°C d e c , [ a ] 2 2 + 244.7° (C 0.76 H 20) . Anal. Calcd. f o r C 1 3H 1 8CuN0 7: C 42.90, H 4.95, N 3.85; found: C 43.50, H 4.82, N 3.68, ms (low r e s o l u t i o n ) c a l c d . f o r C 1 3H 1 8CuN0 7: 363 ( C u 6 3 ) , 365 ( C u 6 5 ) amu (M:M+2; 10:4); found: 363, 365^420 amu. Bis-{N-methyl-3,4,6-tri-0_-acetyl-2-deoxy-2- ( 3 - c a r b o x y l - s a l i c y l -aldimino)} Cu 2 ( I I ) [23] was obtained i n 80% y i e l d , m.p. > 270°C, [ a ] 2 2 + 100.0° (C 0.16 CHC£ 3). Anal, c a l c d . f o r C i + 2 H i + 6 C u 2 N 2 0 2 2 : C 47.67, H 4.38,N 2.65; found: C 47.13, H 4.51, N 2.64; ms (low r e s o l u t i o n ) c a l c d . f o r C i t 2 H l t 6 C u 2 N 2 0 2 2 : 1056 ( C u 6 3 C u 6 3 ) , 1058 ( C u 6 3 Cu 6 5) , 1060 ( C u 6 5 Cu 6 5) amu (M:M+l:M+2:M+3:M+4:M+5:M+6; 100:46:96:39:27:8:2); found: 1056, 1058, 1060 amu (M:M+1:M+2:M+3:M+4:M+5:M+6; 100:50:95:40:30:10:2). P r e p a r a t i o n of methyl-2- (p_-acetamido-benzenesulphonamido)-3,4,6-tri-0-acetyl-2-deoxy-8-D-glucopyranoside [25]. To a s o l u t i o n of the hydrobromide [7] (0.5 g, 1.25 m mol) and sodium bicarbonate (0.26 g, 3 m mol) i n H 20 (5 ml) was added a s o l u t i o n of the sulphonyl c h l o r i d e [24] (0.27 g, 1.17 m mol) i n acetone (5 ml). A f t e r 1 h at room temperature the mixture was evaporated to dryness and chromatographed on s i l i c a g e l using solvent (A) to give the product i n 50% y i e l d from ethanol, m.p. 184-185°C, [ a ] 2 2 - 35.50° (C 1.55 acetone). Ana l , c a l c d . f o r C21H.28N20! XS: C 48.86, H 5.42, N 5.42; found: C 48.88, H 5.48, N 5.31. P r e p a r a t i o n of methyl-2-(p-acetamidobenzene-sulphonamido)-2-deoxy-B-D-glucopyranoside [26]. To a s o l u t i o n of compound [25] (0.2 g, 0.39 m mol) i n dry methanol (2 ml) was added a s o l u t i o n of (0.2 N) sodium methoxide (0.7 ml). The mixture was s t i r r e d at room temperature f o r 20 min and then n e u t r a l i z e d w i t h IR 120 H + i o n exchange r e s i n . The mixture was then f i l t e r e d and evaporated to dryness to give the compound i n 75% y i e l d from ethanol. Anal, c a l c d . f o r C ^ K ^ n ^ O e S : C 46.15, H 5.64, N 7.18; found: C 45.84, H 5.62, N 7.00. P r e p a r a t i o n of methyl-2-(p-amino-benzensulphonamido)-2-deoxy-B-D-glucopyranoside [27]. Compound [26] (0.1 g, 0.26 m mol) was d i s s o l v e d i n NaOH (IN) (10 ml) and heated under r e f l u x f o r 1.25 h. The mixture was then n e u t r a l -i z e d w i t h IR 120 H + i o n exchange r e s i n and the r e s i n then f i l t e r e d and the s o l u t i o n evaporated to dryness. The product was c r y s t a l l i z e d from ethanol to give a y i e l d of 60%, m.p. 201-203°C. Anal, c a l c d . f o r C 1 3H 2o N 20 7S: C 44.83, H 5.75, N 8.04; found: C 44.50, H 5.82, N 7.86. P r e p a r a t i o n of methyl-2-deoxy-2-(p-salicylaldiminobenzenesulphon-amido)-B-D-glucopyranoside [28]. Compound [27] (0.1 g, 0.29 m mol) was d i s s o l v e d i n H 20 (2 ml) and 198 to t h i s s t i r r e d s o l u t i o n at ambient temperature, was added s a l i c y l a l d e -hyde (0.05 g, 0.4 m mol). The mixture was s t i r r e d v i g o r o u s l y f o r 3 h and the product f i l t e r e d to give a 78% y i e l d . The compound was r e c r y s -t a l l i z e d from e t h a n o l , m.p. 193-194°C, M^2 - 55.07 (C 1.42 methanol). Anal , c a l c d . f o r C 2 0H 24N 20 8S: C 53.10, H 5.31, N 6.19; found: C 53.01, H 5.37, N 6.00. VE: Chapter I I ( i ) Sources of M a t e r i a l s Ferrocene was obtained from Sigma Chemical Company, Saint L o u i s , M i s s o u r i . A l l the ferrocene-derived s t a r t i n g m a t e r i a l s were synthesized from known methods (see t e x t ) except f o r 1,1'-ferrocene d i c a r b o x y l i c a c i d which was purchased from Strem Chemicals, Inc., Newburyport, (MA). Sodium hydride was purchased from A l f a products (Danvers, MA) as 50% d i s p e r s i o n i n o i l . p - T o l u e n e s u l f o n y l c h l o r i d e was purchased from Eastman Organic Chemicals (Rochester, N.Y.), and p u r i f i e d by the method of P e l l e t i e r " ^ . Cyanuric c h l o r i d e (97%) was purchased from A l d r i c h Chemical Co., (Milwaukee, Wisconsin). n - b u t y l l i t h i u m (1.6 M) was purchased from A l d r i c h . ( i i ) . L i t e r a t u r e P reparations The mono-ferrocene c a r b o x y l i c a c i d was prepared from m o n o l i t h i o -12 ferrocene by the r e a c t i o n of Goldberg et a l . . Under anhydrous condi-t i o n s n - b u t y l l i t h i u m , i n equimolar amounts, was added to s o l u t i o n of ferrocene i n ether. Excess amounts of n - b u t y l l i t h i u m apparently cause r e a c t i o n at both c y c l o p e n t a d i e n y l r i n g s and using an equimolar amount prevents t h i s mixture. A f t e r 6 h the e t h e r e a l s o l u t i o n was poured i n t o a s l u s h of ether and dry i c e . The e t h e r e a l residue was washed w i t h 199 water and the aqueous e x t r a c t s a c i d i f i e d w i t h 6 N h y d r o c h l o r i c a c i d . A f t e r f i l t r a t i o n and d r y i n g , about 25% y i e l d was obtained as reported. The a c i d c h l o r i d e s [2] and [3] derived from the mono-carboxylic a c i d and the d i - 1 , 1 ' - c a r b o x y l i c a c i d r e s p e c t i v e l y , were prepared by the method 13 of Pauson . To a benzene s o l u t i o n of the a c i d , under a n i t r o g e n atmos-phere, was added an equimolar amount of P C J I 5 and the mixture allowed to s t i r at room temperature f o r 3 h. The benzene s o l u t i o n was then washed w i t h d i l u t e sodium hydroxide and water, and the benzene l a y e r then d r i e d and evaporated. The products can be c r y s t a l l i z e d from pentane but the crude a c i d c h l o r i d e s were always used d i r e c t l y without p u r i f i c a t i o n . The y i e l d s were approximately 60%. The N,N-Dimethylaminomethylferrocene methiodide [12] was prepared 14 by the method of Lednicer . Under a N 2 atmosphere, ferrocene was added to a s t i r r e d s o l u t i o n of b i s (dimethylamino)-methane (prepared by a r e c i p e w i t h i n the same reference) and phosphoric a c i d i n a c e t i c a c i d . The mixture was then heated on a steam bath f o r 5 h. The dark amber mixture was allowed to c o o l and was d i l u t e d w i t h H 20. Any unreacted ferrocene was removed by e x t r a c t i o n w i t h ether. The aqueous s o l u t i o n was then made a l k a l i n e and the t e r t i a r y amine separates as a dark o i l . The mixture was ex t r a c t e d w i t h ether to give the t e r t i a r y amine as a dark red o i l . To t h i s o i l , d i s s o l v e d i n methanol, was added methyl i o d i d e . The s o l u t i o n was then heated f o r 5 min and a f t e r c o o l i n g , ether was added. The r e s u l t i n g p r e c i p i t a t e was f i l t e r e d to give the product i n a high y i e l d . Since t h i s reagent i s water s o l u b l e i t can be e a s i l y removed from r e a c t i o n mixtures by e x t r a c t i o n . The a l c o h o l [18] was e a s i l y prepared from the above reagent [12] by simply b o i l i n g the methiodide i n sodium hydroxide s o l u t i o n f o r 200 3.5 h ^ . The product i s then i s o l a t e d by e x t r a c t i o n w i t h ether. Trimethylamine i s l i b e r a t e d and ther e f o r e the r e a c t i o n must be done i n the fume hood. Holding a moistened piece of pH paper over the top of the condensor i s a convenient way of determining the r e a c t i o n time. The ferrocene carboxaldehyde [24] was a l s o prepared from the 16 methiodide [12] by the method of Broadhead et a l . . The methiodide [12], hexamethylenetetramine and a c e t i c a c i d were heated under r e f l u x f o r 1 min and then the mixture was poured i n t o water and ext r a c t e d w i t h benzene to give the product i n 27% y i e l d as compared to the l i t e r a t u r e v alue of 37%. The aldehyde was r e c r y s t a l l i z e d from 25% ethanol/water. ( i i i ) Synthesis of Ferrocenyl-Sugar Conjugates Reactions of 1-ferrocenecarbonyl c h l o r i d e [2] and of l , l ' - f e r r o c e n e d i c a r b o n y l c h l o r i d e [3] w i t h sugars [ 4 ] , [ 5 ] , and [10]. E i t h e r 1 or 2 eq u i v a l e n t s of the t h i o [4] or amino [5] sugars were added to a s t i r r e d s o l u t i o n of [2] or [3] (0.25 g) i n dry chloroform (15 ml). Then 1 or 2 equivalents of e i t h e r p y r i d i n e or t r i e t h y l a m i n e was added and the r e a c t i o n allowed to s t i r f o r 15 min at room temperature. The mixture was then extracted twice w i t h each o f : water, 5% sodium carbonate, and water again. The chloroform l a y e r was d r i e d over anhyd-rous sodium s u l f a t e , f i l t e r e d and evaporated to dryness. The l , 2 : 5 , 6 - d i -CHisoproprylidene glucofuranose sugar [10] was reacted w i t h [2] i n dry p y r i d i n e overnight and a f t e r c o n c e n t r a t i o n , chromatographed on a s i l i c a g e l column using solvent (B). 2,3,4,6-Tetra-O-acetyl-l-deoxy-l-S-(1-ferrocenecarboxylate)-B-D-glucopyranose [6] was obtained i n 60% y i e l d from ethanol, m.p. 186-187°C, [ a ] 2 2 + 35.0° (C 1 CHC£ 3). Anal, c a l c d . f o r 0 2 5 ^ 8 ^ 0 ! 0 S : C 52.11, H 4.86, S 5.56; found C 52.02, H 4.92, S 5.80. 201 1,1'-bis-(S-(2,3,4,6-tetra-0-acetyl-l-deoxy-l-thio-8-D-glucopyran-ose)} ferrocenecarboxylate [7] was obtained i n 56% y i e l d from e t h a n o l , m.p. 213-214°C, [ a ] 2 2 - 36.0° (C 1 CHC£ 3). Anal, c a l c d . f o r C i ^ H ^ F e O 2 0S 2: C 49.71, H 4.76, S 6.64; found: C 49.56, H 4.76, S 6.55. {2-N-(1,3,4,6-tetra-0-acetyl-2-amino-2-deoxy-6-D-glucopyranose)}-1-ferrocenecarboxamide [8] was obtained i n 50% y i e l d from ethanol, m.p. 182-183°C, [ a ] 2 2 - 2.5° (C 1 CHCJ>3). Anal, c a l c d . f o r C 2s^gFeNO! 0 : C 53.70, H 5.19, N 2.50; found: C 54.00, H 5.23, N 2.51. Bis-{2-N-(1,3,4,6-tetra-0-acetyl-2-amino-2-deoxy-8-D-glucopyranose)} -1,1-ferrocenecarboxamide [9] was obtained i n 60% y i e l d from e t h a n o l , m.p. 110-110°C, [ a ] 2 2 + 20.0° (C 1 CHC£ 3). Anal , c a l c d . f o r Ct t 0Hi + 8FeN 2O 2 0: C 51.53, H 5.15, N 3.00; found: C 51.29, H 5.25, N 2.98. {3-0-(1,2:5,6-di-0-isopropylidene-a-D-glucofuranose)}-l-ferrocene carboxylate [11] was prepared i n 50% y i e l d from column chromatography using solvent (B) and c r y s t a l l i z a t i o n from ethanol/H 20, m.p. 110-111°C, [ a ] 2 2 - 55.5° (C 0.64 CHC£ 3). Anal, c a l c d . f o r C 2 2 H 2 8 F e 0 5 : C 59.50, H. 6.31; found: C 59.27, H 6.07. Pr e p a r a t i o n of 1,3,4,6-tetra-0-acetyl-2-N,N-bis-(1-aminomethyl-ferrocene)-2-deoxy-8-D-glucopyranose [14]. A mixture of N,N-dimethylaminomethylferrocene methiodide (2.5 g, -3 -3 6.5 x 10 mol), the 2-amino sugar [5] (0.9 g, 2.6 x 10 mol) and sodium _3 carbonate (0.8 g, 7.5 x 10 mol) i n a c e t o n i t r i l e (40 ml) was heated to r e f l u x f o r 12 h. The mixture was then evaporated to dryness, d i s s o l v e d i n chloroform and ext r a c t e d f i v e times w i t h water. The chloroform 202 l a y e r was d r i e d over anhydrous sodium s u l f a t e , f i l t e r e d , evaporated to dryness and a f t e r t r i t u r a t i o n w i t h hexane, f i l t e r e d to give 1.28 g (66%). The compound was r e c r y s t a l l i z e d by d i s s o l u t i o n i n a minimum of benzene, followed by a d d i t i o n of a small amount of hexane and, subsequent a d d i t i o n of petroleum ether (bp 30-60°C) u n t i l a s l i g h t l y cloudy s o l u t i o n was obtained and then c o o l i n g ; m.p. 97-98°C, [ a ] 2 2 + 77.0° (C 1 CHC£ 3). Anal, c a l c d . f o r C 3 5H 1 + 1Fe 2N0 9: C 58.19, H 5.52, N 1.88; found: C 57.88, H 5.56, N 1.93. P r e p a r a t i o n of 2 , 3 , 4 , 6 - t e t r a - 0 - a c e t y l - l - S - ( 1 - t h i o m e t h y l f e r r o c e n e ) -3-D-glucopyranose [13] from [12]. A mixture of 2,3,4,6-tetra-0-acetyl-l-thio-B-D-glucopyranose (2 g, -3 -3 5.5 x 10 mol), the methiodide [12] (2 g, 5.2 x 10 mol) and anhydrous -3 sodium carbonate (0.6 g, 5.6 x 10 mol) i n a c e t o n i t r i l e (50 ml) was r e f l u x e d f o r 12 h. The mixture was evaporated to dryness, d i s s o l v e d i n chloroform and e x t r a c t e d f i v e times w i t h water. The chloroform l a y e r was d r i e d over anhydrous sodium s u l f a t e , f i l t e r e d and evaporated to dryness. The product was then column chromatographed on s i l i c a g e l using solvent (A) and c r y s t a l l i z e d from ethanol to give 0.5 g (17%), of [13], m.p. 129-130°C, [ a ] 2 2 + 32.0° (C 0.85 CHC£ 3). This product however was found by !H nmr to be a mixture of a- and 3- anomers (40:60). F i v e repeated r e c r y s t a l l i z a t i o n s from ethanol gave the pure 3 anomer m.p. 139-140°C, [ a ] 2 2 - 39.0° (C 1 CHC£ 3). Anal, c a l c d . f o r C 25H 3 0FeO 9S: C 53.41, H 5.34, S 5.70; found: C 53.21, H 5.34, S 5.93. 203 P r e p a r a t i o n of (1-hydroxymethylferrocene)p-toluenesulphonyl c h l o r i d e [16]. -3 Sodium hydride (0.12 g, 2.5 x 10 mol) was placed i n a 50 ml, two necked f l a s k and washed twice w i t h anhydrous ether. Then dry ether was _3 added (15 ml) along w i t h dry 1-hydroxy methyl ferrocene (0.5 g, 2.3 x 10 mol). The mixture was then heated under r e f l u x overnight under n i t r o g e n . The s t i r r e d suspension of the a l c o h o l a t e was cooled to -23°C and to t h i s was added dropwise, over a period of 0.5 h, a s o l u t i o n of p-toluene--3 sulphonyl c h l o r i d e (0.44 g, 2.3 x 10 mol), i n anhydrous ether (15 ml). The mixture was s t i r r e d at -10°C f o r two more hours and allowed to warm to room temperature f o r another hour. The mixture was then f i l t e r e d under n i t r o g e n through s i n t e r e d g l a ss and the r e s u l t i n g s o l u t i o n of the t o s y l a t e [16] used d i r e c t l y i n subsequent r e a c t i o n s . P r e p a r a t i o n of 2,3,4,6-tetra-C)-acetyl-l-S-(1-thiomethylferrocene)-8-D-glucopyranose [13] using the t o s y l a t e [16]. -3 1-Hydroxymethylferrocene (0.5 g, 2.3 x 10 mol) was converted i n t o the t o s y l a t e [16], to an ether s o l u t i o n of which was added a s o l u t i o n of _3 the 1-thio sugar [4] (0.5 g, 1.4 x 10 mol) i n dry chloroform (10 ml). The mixture was kept f o r 40 h at ambient temperature and was then f i l -t e red and evaporated to dryness. Upon a d d i t i o n of e t h a n o l , the product c r y s t a l l i z e d to give 0.67 g (87%) of [13]. One r e c r y s t a l l i z a t i o n from ethanol gave orange p l a t e s i n 79% y i e l d , m.p. 139-140°C. For f u r t h e r a n a l y t i c a l data see the p r e p a r a t i o n v i a the methiodide [12]. 204 P r e p a r a t i o n of 1,3,4,6-tetra-C)-acetyl-2-N-(1-aminomethylferrocene)-6-D-glucopyranose [15]. -3 1-Hydroxymethylferrocene (0.5 g, 2.3 x 10 mol) was converted i n t o the t o s y l a t e ; to an ether s o l u t i o n of the t o s y l a t e was added a s o l u t i o n of the 2-amino sugar [5] i n dry chloroform (20 ml). The mixture was heated under r e f l u x f o r two days and was then f i l t e r e d , evaporated to dryness, d i s s o l v e d i n ether and e x t r a c t e d i n t o 5% aqueous HC£. The acqueous l a y e r was then n e u t r a l i z e d w i t h sodium bicarbonate and e x t r a c t e d w i t h ether. The ether l a y e r was d r i e d over anhydrous sodium s u l f a t e , evapor-ated to a s m a l l volume and cooled. The compound [15] c r y s t a l l i z e d as f i n e needles i n 30% y i e l d , m.p. 126-127°C, [ a ] 2 2 + 8.0° (C 1 CHC& 3). Anal, c a l c d . f o r C25H3iFeN0g: C 55.08, H 5.69, N 2.57; found: C 54.79, H 5.54, N 2.69. P r e p a r a t i o n of 1,2:3,4-di-0-isopropylidene-3-0-(1-hydroxymethyl-ferrocene)-a-D-galactopyranose [20]. -3 1-Hydroxymethylferrocene (0.5 g, 2.3 x 10 mol) was converted i n t o the t o s y l a t e and to the ether s o l u t i o n was added the 6-hydroxy sugar [19] _3 (0.45 g, 1.7 x 10 mol) i n anhydrous ether (5 ml). The mixture was kept f o r two days and was then f i l t e r e d , evaporated to dryness and p u r i f i e d on s i l i c a g e l column chromatography using solvent (A), to give the compound as an o i l i n 20% y i e l d , [ a ] 2 2 - 58.7° (C 0.8 CHC&3). Anal , c a l c d . f o r C23H3QFe06: C 60.30, H 6.55; found: C 60.16, H 6.59. 205 P r e p a r a t i o n of 2-deoxy-2-N-(1-ferrocenecarboxaldehydeimine)-a,6-D-glucopyranose [26]. -3 A mixture of 1-ferrocenecarboxaldehyde (0.5 g, 2.3 x 10 mol), -3 2-amino-2-deoxy-a, g-D-glucopyranose hydr o c h l o r i d e (0.5 g, 2.3 x 10 -3 mol) and t r i e t h y l a m i n e (0.24 g, 0.24 x 10 mol) i n absolute ethanol (15 ml) was s t i r r e d v i g o r o u s l y f o r 15 h. The mixture was then evapor-ated to a small volume and an equal amount of acetone added. The mixture was cooled and f i l t e r e d to give 0.68 g (71%) product which was r e c r y s t a l -l i z e d by d i s s o l v i n g i n a minimum of ethanol, evaporating to about h a l f i t s volume, adding hexane and r e f r i g e r a t i o n ; m.p. 161-162°C, [ a ] ^ 2 + 66.7 C (C 0.33 MeOH). Anal, c a l c d . f o r C 1 7 H 2 i F e N 0 5 : C 54.39, H 5.64, N 3.73; found: C 54.09, H 5.90, N 3.90. Pr e p a r a t i o n of 2,4-Dichloro-6-(1-hydroxymethylferrocene)-s-t r i a z i n e [22]. To an i c e co l d s t i r r e d s o l u t i o n of cyanuric c h l o r i d e (0.86 g, -3 4.6 x 10 mol) i n acetone (20 ml), over a pe r i o d of 45 minutes was added, simultaneously, a mixture of hydroxymethyl ferrocene (1 g, _3 4.6 x 10 mol) i n acetone (40 ml) and a s o l u t i o n of sodium hydroxide (5 ml of 4%) i n water (55 ml). The s o l u t i o n was then f i l t e r e d to y i e l d a compound which was i d e n t i f i e d as the ferrocene ether dimer. The f i l -t r a t e was allowed to become a c i d i c upon standing at 0°C and the r e s u l t -i n g orange p r e c i p i t a t e f i l t e r e d a f t e r 2 h to give the compound i n 50% y i e l d . This product i s s u f f i c i e n t l y pure f o r f u r t h e r r e a c t i o n s and should be stored i n an evacuated d e s i c c a t o r . P u r i f i c a t i o n was achieved by column chromatography on s i l i c a g e l using solvent (A) and c r y s t a l l i z e d from ether/hexane, m.p. 120°C (Dec). A n a l , c a l c d . f o r C1^1C£2FeN3°: 206 C46.21, H 3.02, N 11.54; found: C 46.68, H 3.18, N 11.24; ms (high reso-l u t i o n ) c a l c d . f o r C 1 J tH nCJl2FeI>i30: 362.9615 amu; found 362.9625. Bis-{l-hydroxymethylferrocene}-ether was obtained i n 15% y i e l d , m.p. 113-114°C. Anal, c a l c d . f o r C22H 2 2Fe 20: C 63.83, H 5.32; found: C 63.84, H 5.45; ms (low r e s o l u t i o n ) c a l c d . f o r C 22H22F e20 ; 414 amu; found: 414. Prepa r a t i o n of 2- (1-hydroxymethylf errocene)-4,6-bis-S_- (2, 3,4 ,6-t e t r a - 0 - a c e t y l l - d e o x y - l - t h i o - 8 - D - g l u c o p y r a n o s e ) - s - t r i a z i n e [23]. -3 Triethylamine (0.2 g, 2.0 x 10 mol) i n a c e t o n i t r i l e (2 ml) was -3 added to a s t i r r e d s o l u t i o n of compound [22] (0.3 g, 8.3 x 10 mol) and _3 1-thio glucose [4] (0.6 g, 1.65 x 10 mol) i n a c e t o n i t r i l e (25 ml). A f t e r 10 min the mixture was poured i n t o i c e water (100 ml) and ext r a c t e d w i t h chloroform. The chloroform l a y e r was d r i e d over anhydrous sodium s u l f a t e , f i l t e r e d and evaporated to dryness. One r e c r y s t a l l i z a t i o n from isopropanol gave 0.6 g (74%), m.p. 120-121°C, [ a ] 2 2 + 25.4 (C 1 CHC£ 3). Anal, c a l c d . f o r Ci + 2 H i + 9 F e N 3 0 1 9 S 2 : C 49.48, H 4.81, N 4.12; found: C 49.51, H 4.72, N 4.12; ms (high r e s o l u t i o n ) c a l c d . f o r C i + 2 H 1 + 9 F e N 3 0 i 9 S 2 : 1019.1749 amu; found: 1019.1769. P r e p a r a t i o n of 1-deoxy-l-S-(1-thiomethylferrocene)-g-D-gluco-pyranose [27]. To a s t i r r e d s o l u t i o n of compound [13] (0.1 g) i n methanol/chloro-form (4 ml 1:1) was added dry 0.2 N sodium methoxide (0.3 ml). A f t e r 30 min under n i t r o g e n , the s o l u t i o n was n e u t r a l i z e d w i t h IR 120 H + ion exchange r e s i n , f i l t e r e d , evaporated to dryness and c r y s t a l l i z e d from H 20 to give 52 mg (75%). R e c r y s t a l l i z a t i o n from H 20 gave orange p l a t e s of the monohydrate, m.p. 145-146°C, [ a ] 2 2 - 43.2° (C 0.44 MeOH). Anal. 207 c a l c d . f o r C i 7 H 2 2 F e 0 5 S : C 49.55, H 5.82, S 7.78; found: C 49.84, H 5.65, S 7.70. P r e p a r a t i o n of 1-deoxy-l-S-(1-ferrocenecarboxylate)-B-D-gluco-pyranose [28]. To a s t i r r e d s o l u t i o n of the t h i o e s t e r [6] (0.1 g) i n methanol/ chloroform (4 ml, 1:1) was added 0.2 N sodium methoxide (0.7 ml) and the s o l u t i o n was s t i r r e d f o r 45 min under n i t r o g e n . The s o l u t i o n was then n e u t r a l i z e d w i t h IR 120 H + r e s i n and the mixture f i l t e r e d , evaporated to dryness and the compound chromatographed on a s i l i c a g e l column using solvent (C) to give 35 mg (50%) from e t h a n o l , m.p. 145-146°C, [ a ] 2 2 - 3.3° (C 0.3 CHC£ 3). Anal , c a l c d . f o r C 1 7H 2 0FeO 6S: C 50.03, H 4.90; found: C 49.75, H 5.08. VF: Chapter I I I ( i ) Sources of M a t e r i a l s C e l l u l o s e powder (Watman CF11) was obtained as a g i f t from Drs. S. Chow, P. R. St e i n e r and J . N. R. Ruddick. Agarose (SK-ME 11335) was obtained as a g i f t from Marine C o l l o i d s . I t was a white granular s o l i d c o n t a i n i n g £ 0.5% methoxyl, 0.2-3% pyruvate, 0.65% ash, 0.28% s u l f a t e and had a molecular weight ' v 10 5. Sephadex G25 medium was purchased from Pharmacia. Both xanthan and guar gum were g i f t s from Kelco (San Diego, C a l . ) , s t a r c h was obtained from A l l i e d Chemical Co. The s p i n l a b e l 4-amino-2,2,6,6-tetramethylpiperidine-2-oxyl [10], n-hexylamine, and n-dodecylamine were purchased from Eastman. Cyanuric c h l o r i d e was purchased from A l d r i c h . 1,2:5,6-di-O-isopropylidine-a-D-glucopyranose [28] was purchased from Koch-Light L a b o r a t o r i e s L t d . 208 (Coinbrook Bucks, England). The 1-thio-glucose [31] had p r e v i o u s l y been prepared by a standard recipe"'"''. p-Toluidine chromium t r i c a r b o x y l was purchased from Strem (Newburyport, MA, USA). 2,2,6,6-tetramethyl-4-p i p e r i d i n o l was purchased from A l d r i c h . ( i i ) L i t e r a t u r e Preparations 4-hydroxy-2,2,6,6-tetramethylpiperidine-l-oxyl [11] was prepared by 18 the method of Rozantsev by o x i d i z i n g the secondary amine precursor. 2 , 2 , 6 , 6 - t e t r a m e t h y l - 4 - p i p e r i d i n o l was mixed w i t h hydrogen peroxide, EDTA, and sodium tungstate i n water f o r f i v e days. Then the mixture was saturated w i t h potassium carbonate and e x t r a c t e d w i t h ether. The r e s i d u e a f t e r evaporating was r e c r y s t a l l i z e d to give l a r g e orange c r y s t a l s of the f r e e r a d i c a l [11] i n high y i e l d . P u r i t y can be checked e a s i l y by t i c using methanol or a t o l u e n e / e t h y l acetate 1:1 mixture. One r e c r y s -t a l l i z a t i o n from ether-hexane (2:1) gives the pure compound. The p r e p a r a t i o n of 4- [2, 4 - D i c h l o r o - s _ - t r i a z i n e - 6 - y l ) - 2 , 2 ,6,6-19 t e t r a m e t h y l a m i n o p i p e r i d i n - l - o x y l [12] has been made before but the present method described i s f a r more convenient. Cyanuric c h l o r i d e (0.54 g, 2.9 x 10 mol) was added to acetone (12 ml) and s t i r r e d at 0°C. To t h i s s t i r r e d s o l u t i o n was added an aqueous s o l u t i o n (20 ml) of 4-amino--3 2 , 2 , 6 , 6 - t e t r a m e t h y l - p i p e r i d i n e - l - o x y l (0.5 g, 2.9 x 10 mol) and sodium -3 bicarbonate (0.24 g, 2.9 x 10 mol). The mixture was then s t i r r e d at 0°C f o r l 1 h and then f i l t e r e d and washed w i t h c o l d water. The product was heated under vacuum to 50°C i n a s u b l i m a t i o n apparatus (to remove unreacted cyanuric c h l o r i d e ) , g i v i n g a 75% y i e l d , m.p. 195°C. Anal, c a l c d . f o r C 1 2H 1 8CK. 2N 50 1 : C 45.17, H 5.65, N 21.96; found: C 45.38, H 5.79, N 21.70. 209 ( i i i ) P o lysaccharide and Surface D e r i v a t i z a t i o n Spin l a b e l l i n g of c e l l u l o s e , agarose, and Sephadex G25, v i a cyanuric c h l o r i d e was achieved by the f o l l o w i n g method. The i n s o l u b l e polysaccharide (0.2 g) was mixed w i t h NaOH (8%) f o r 1 h and then the excess base was decanted and the wet polysaccharide allowed to stand at room temperature overnight. Then a s o l u t i o n of one of the t r i a z i n e s p i n l a b e l s [ 1 2 ] ) , [22] or [26] (0.2 g) i n aqueous acetone (1:3) (3 ml) was added and the mixture shaken f o r 1 h. The products were then f i l t e r e d on a s i n t e r e d funnel and washed with water. The products were then shaken i n water overnight to remove any tra c e s of unreacted l a b e l and then f i l t e r e d again. A f t e r f i l t r a t i o n , c e l l u l o s e and agarose were f i r s t washed w i t h methanol and then ether, to remove water, whereas the Sephadex beads were f i r s t washed w i t h ethanol and then ether. The products were d r i e d i n a d r y i n g p i s t o l under 0.01 t o r r pressure at 56°C. Spin l a b e l l i n g of the water s o l u b l e polysaccharides xanthan gum, guar gum, and s t a r c h was performed i n the f o l l o w i n g way. The pol y -saccharide (0.2 g) was d i s s o l v e d i n 4% NaOH (5 ml) and to t h i s s o l u t i o n was added an acetone s o l u t i o n (3 ml) of the s p i n l a b e l . The mixtures were then shaken f o r 1 h at room temperature. Guar and xanthan gum were p r e c i p i t a t e d by adding acetone (^  20 ml) and were then f i l t e r e d and washed w i t h acetone. The s t a r c h sample was p u r i f i e d by gel f i l t r a t i o n u s i n g Sephadex LH-20. L a b e l l i n g of Bovine Serum Albumin (BSA) was achieved by the f o l l o w -i n g method. BSA (0.02 g) was d i s s o l v e d i n phosphate b u f f e r (pH 8, 1 ml) and to t h i s s o l u t i o n was added an ethanol s o l u t i o n (0.2 ml) of the s p i n l a b e l [22] (10 mg). The mixture was then l e f t overnight at room 210 temperature and was p u r i f i e d by g e l f i l t r a t i o n u s i n g Sephadex G25 el u t e d w i t h pH 7 phosphate b u f f e r . Uv absorption at 280 nm was used to id e n -t i f y the p r o t e i n c o n t a i n i n g f r a c t i o n s . The f r a c t i o n s were then concen-t r a t e d u s i n g dry Sephadex beads before esr spectra were run. Aluminum Oxide ( b a s i c , Brockman a c t i v i t y 1) was l a b e l l e d very e a s i l y by the f o l l o w i n g procedure. A mixture of alumina (1 g) and the s p i n l a b e l [22] (0.1 g) were mixed i n chloroform f o r 15 min and was then f i l t e r e d on a s i n t e r e d f u n n e l . The m a t e r i a l was washed w i t h chloroform and methanol and was then shaken i n chloroform overnight. A f t e r t h i s , the m a t e r i a l was f i l t e r e d again and d r i e d i n the d r y i n g p i s t o l under 0.01 t o r r pressure at 56°C. C o n t r o l l e d pore g l a s s was l a b e l l e d w i t h reagent [22] by Dr. J . C. Waterton w h i l e working at UBC, usin g the f o l l o w i n g procedure. 3-amino 20 p r o p y l c o n t r o l l e d pore g l a s s was prepared by mixing the c o n t r o l l e d pore glass w i t h t r i - e t h o x y - p r o p y l a m i n e - s i l o x a n e i n water. The glass beads were then f i l t e r e d . To a dry sample of t h i s m a t e r i a l (11.6 mg) was added the l a b e l [22] (6.3 mg) i n a c e t o n i t r i l e (5 ml) w i t h t r i e t h y l a m i n e (1 ml). The mixture was shaken f o r 24 h at room temperature and washed w i t h a c e t o n i t r i l e and d r i e d at 110°C/0.01 t o r r f o r 24 h. The y i e l d was 18% based on the l e v e l of propylamine m o d i f i c a t i o n , ( i v ) Synthesis of s_-Triazine Compounds Pr e p a r a t i o n of 4 - [ 2 - c h l o r o - 4 ( n - h e x y l a m i n o ) - s - t r i a z i n - 6 - y l ] -2,2,6,6-tetramethylaminopiperidin-l-oxyl [13]. -4 Compound [12] (0.2 g, 6.3 x 10 mol) was d i s s o l v e d i n acetone (5 ml) and added w i t h s t i r r i n g to water (5 ml) at 0°C. To t h i s s t i r r e d s o l u t i o n was added an aqueous s o l u t i o n (5 ml) of sodium bicarbonate (0.06 g, 7 x 10 4 mol) and an acetone s o l u t i o n (5 ml) of n-hexylamine -4 (0.064 g, 6.3 x 10 mol). The mixture was then s t i r r e d f o r 20 min at 45°C and the product f i l t e r e d , washed w i t h c o l d water and d r i e d to y i e l d 0.24 g (83%) from ethanol/water, m.p. 160-161°C. Anal, c a l c d . f o r C 1 8H 3 2C£iN 60 1: C 56.32, H 8.34, N 21.90; found: C 56.34, H 8.21, N 21.90. Pr e p a r a t i o n of 4- [2-chloro-4- ( n - d o d e c y l a m i n o ) - s - t r i a z i n - 6 - y l ] -2,2,6,6-tetramethylaminopiperidin-l-oxyl [14]. -4 Compound [12] (0.25 g, 7.8 x 10 mol) was d i s s o l v e d i n acetone (10 ml) and added w i t h s t i r r i n g to water (7 ml) at 0°C. To t h i s s t i r r e d s o l u t i o n was added an aqueous s o l u t i o n (10 ml) of sodium bicarbonate -4 (0.07 g, 8.3 x 10 mol), and a hot acetone s o l u t i o n (10 ml) of dodecyl-amine (0.145 g, 7.8 x 10 ^  mol). The mixture was s t i r r e d at 45°C f o r 40 min and the pink compound was then f i l t e r e d , washed w i t h water and d r i e d . The compound required no f u r t h e r p u r i f i c a t i o n and was obtained i n 82% y i e l d , m.p. 118-119°C. Anal, c a l c d . f o r 021^1, CJ^NeOi : C 61.63, H 9.41,N 17.96; found: C 61.66, H 9.41, N 18.16. Pr e p a r a t i o n of 4-[4,6-di-(n-dodecylamino)-s-triazin-6-yl]-2,2,6,6-t e t r a m e t h y l a m i n o p i p e r i d i n - l - o x y l [15]. -4 Compound [14] (0.2 g, 4.3 x 10 mol) xvas d i s s o l v e d i n acetone (5 ml) and added w i t h s t i r r i n g to water (5 ml). To t h i s s t i r r e d s o l u -t i o n was added an aqueous s o l u t i o n (5 ml) of sodium bicarbonate (0.04 g, 4.8 x 10 ^  mol) and a hot acetone s o l u t i o n (10 ml) of dodecylamine (0.08 g, 4.3 x 10 4 mol). The mixture was s t i r r e d overnight at 80°C and the pink compound was then f i l t e r e d , washed w i t h water and d r i e d . The product needed no f u r t h e r p u r i f i c a t i o n and was obtained i n 85% y i e l d , m.p. 78-79°C. Anal, c a l c d . f o r C3&E70^701: C 70.15, H 11.36, N 15.90; 212 found: C 70.46, H 11.20, N 15.74. Pr e p a r a t i o n of 2 , 4 - D i c h l o r o - s _ - t r i a z i n - 6 - p - t o l u i d i n e chromium t r i c a r b o n y l [17]. -3 Cyanuric c h l o r i d e (0.4 g, 2.2 * 10 mol) was d i s s o l v e d i n acetone (8 ml) at 0°C and to t h i s s t i r r e d s o l u t i o n was added an acetone s o l u t i o n -3 (7 ml) of p - t o l u i d i n e chromium t r i c a r b o n y l (0.53 g, 2.2 x 10 mol) and -3 an aqueous s o l u t i o n (45 ml) of sodium bicarbonate (0.3 g, 3.6 x 10 mol). The mixture was s t i r r e d at 0°C f o r 2 h and f i l t e r e d to give a y i e l d of 85%. The product was r e c r y s t a l l i z e d from ethanol/hexane, m.p. • 160-161°C (dec), i r (C=0 s t r e t c h ) 1950 and 1850 cm"1. Anal, c a l c d . f o r ClsHeCi^CrxNi+Os: C 39.93, H 2.05, N 14.32; found: C 40.26, H 2.22, N 14.06. Pr e p a r a t i o n of 2 - ( 2 , 4 - D i c h l o r o - s - t r i a z i n - 6 - y l ) m e t h y l - 3 , 4 , 6 - t r i -0-acetyl-2-amino-2-deoxy-glucopyranose [21]. -4 Cyanuric c h l o r i d e (0.12 g, 6.5 x 10 mol) was d i s s o l v e d i n acetone (3 ml) and to t h i s i c e cold s t i r r e d s o l u t i o n was added an aqueous s o l u -t i o n (8 ml) of methyl-3,4,6-tri-0-acetyl-2-amino-2-deoxy-glucopyranoside hydrobromide (0.25 g, 6.3 x 10 4 mol) and sodium bicarbonate (0.12 g, _3 1.4 x 10 mol). The mixture was s t i r r e d f o r 1 h at 0°C and then f i l t e r e d to give the product i n a 76% y i e l d , m.p. 190-191°C, [ a ] 2 2 - 30.0' (C 1 CHCX,3). Anal, c a l c d . f o r C i 6 H 2 Q C ^ H O Q : C 41.13, H 4.28, N12.00; found: C 41.36, H 4.50, N 11.90. 213 P r e p a r a t i o n of 4-[2 , 4 - D i c h l o r o - s - t r i a z i n - 6 - y l o x y ] - 2 , 2 , 6 , 6 - t e t r a m -e t h y l p i p e r i d i n - l - o x y l [22]. -2 4-hydroxy-2,2,6,6-tetramethylpiperidine-l-oxy (2.0 g, 1.2 x 10 mol) was d i s s o l v e d i n a mixture of water (50 ml) and 4% NaOH (12 ml) and was added dropwise to a s t i r r e d i c e cold acetone s o l u t i o n (40 ml) -3 of cyanuric c h l o r i d e (1.8 g, 9.8 * 10 mol) over a period of 1 h. The orange product was then f i l t e r e d , washed w i t h water and heated to 50°C i n a su b l i m a t i o n apparatus to remove unreacted cyanuric c h l o r i d e . The product r e q u i r e d no f u r t h e r p u r i f i c a t i o n and was obtained i n a 16% y i e l d , m.p. 108-109°C. Anal, c a l c d . f o r C i 2 H i 7 C H 2 ^ 2 : C 45.05, H 5.31, N 17.50; found: C 44.74, H 5.42, N 17.56. Pr e p a r a t i o n of 4-[4-chloro-6-(methyl-3,4,6-tetra-0-acetyl-2-deoxy-6-D-glycopyranosyl) amino-s-triazin-2-yl]-2,2,6,6-tetramethylaminopiper-i d i n - l - o x y l [23]. -4 A mixture of [22] (0.1 g, 3.1 x 10 mol) methy1-3,4,6-tri-0-acetyl-2-amino-2-deoxy-8-p-glycopyranose hydrobromide (0.125 g, -4 -4 3.1 x 10 mol) and sodium carbonate (0.1 g, 9.4 x 10 mol) was s t i r r e d i n a c e t o n i t r i l e (30 ml) at room temperature overnight. The mixture was then f i l t e r e d and the f i l t r a t e evaporated to dryness. The product was then p u r i f i e d s i l i c a column chromatography using solvent (A). The orange compound was c r y s t a l l i z e d from ethanol/water to give a 53% y i e l d , m.p. 90-91°C, [a]J2 - 39.0° (C 1 CHC£ 3). Ana l , c a l c d . f o r C25H37CX.1N5O10: C 49.82, H 6.14, N 11.61; found: C 49.69, H 6.35, N 11.32. 214 P r e p a r a t i o n of 4-[2-chloro-4-(n-hexylamino)-s^-triazin-6-yloxy]-2 , 2 , 6 , 6 - t e t r a m e t h y l p i p e r i d i n - l - o x y l [24]. -4 Compound [12] (0.1 g, 3.2 x 10 mol) was d i s s o l v e d i n acetone (3 ml) and added to an acetone (2 m l ) , water (5 ml) s o l u t i o n of n-hexyl--4 -4 amine (0.032 g, 3.2 x 10 mol) and sodium bicarbonate (0.03g, 3.6 x 10 mol) and- the mixture was then s t i r r e d at room temperature f o r 20 min. The product was e x t r a c t e d w i t h chloroform and the chloroform l a y e r washed w i t h water and d r i e d over sodium s u l f a t e . The orange product was then f i l t e r e d , evaporated to dryness and passed down a short s i l i c a g e l column using solvent (A) to give an orange syrup i n 75% y i e l d . Anal. c a l c d . f o r Ci8H3iCJtiN 50 2: C 56.20, H 8.06, N 18.20: found: C 56.22, H 7.85, N 17.90. P r e p a r a t i o n of 4 , 4 [ 2 - c h l o r o - s - t r i a z i n - 4 , 6 - y l o x y ] - d i - 2 , 2 , 6 , 6 -t e t r a m e t h y l p i p e r i d i n - l - o x y l [25]. -2 4-hydroxy-2,2,6,6-tetramethylpiperidine-l-oxyl (2 g, 1.2 x i o mol) was d i s s o l v e d i n a mixture of H 20 (50 ml) and 4% NaOH (13 ml) and added dropwise, over a p e r i o d of 40 min to an i c e c o l d s t i r r e d acetone -3 s o l u t i o n (40 ml) of cyanuric c h l o r i d e (1 g, 5.4 x 10 mol). The r e a c t i o n was l e f t to s t i r f o r l£ h at room temperature and the orange product then f i l t e r e d and washed w i t h water. The product was then d r i e d at 50°C, to remove any unreacted cyanuric c h l o r i d e , to give the compound i n 10% y i e l d , m.p. 194-195°C. Anal, c a l c d . f o r 02^31+0^501+: C 55.36, H 7.46, N 15.36; found: C 55.10, H 7.31, N 15.08. 215 P r e p a r a t i o n of A - [ 4 - ( 1 , 2 : 3 , 4-di - 0-isopropylidene-galactopyranosyl) o x y - 6 - ( n - h e x y l a m i n o ) - s - t r i a z i n - 2 - y l o x y ] - 2 , 2 , 6 , 6 - t e t r a m e t h y l p i p e r i d i n - l -o x y l [30]. -A A mixture of compound [12] (0.1 g, 3.1 x 10 mol), 1 ,2:3,4-di -0--A isopropylidene-a-D-galactopyranose (0.08 g, 3.1 x 10 mol), and one crushed sodium hydroxide p e l l e t were s t i r r e d at room temperature i n benzene (10 ml) overnight. The mixture was then f i l t e r e d and evapor-ated to dryness. To t h i s syrup was added an i c e c o l d aqueous/acetone -A s o l u t i o n (10 ml 25:75) of sodium bicarbonate (0.03 g, 3.6 x 10 mol) -4 and n-hexylamine (0.03 g, 3 x 10 mol). The mixture was allowed to warm to room temperature and then s t i r r e d f o r an a d d i t i o n a l 2 h. The pink compound was p u r i f i e d by s i l i c a g e l column chromatography using solvent (A) and c r y s t a l l i z e d from ethanol/water to give a A0% o v e r a l l y i e l d , m.p. 170-171°C, [ a ] 2 2 _ 55.0° (C 1.4 CHC£3). Anal , c a l c d . f o r C 3oH5oN 50 8: C 59.23, H 8.22, N 11.51; found: C 59.44, H 8.25, N 11.40. P r e p a r a t i o n of 2 , 4 , 6-tri- ( 2 , 3 , 4 , 6 -tetra - 0-acetyl-8-D-glucopyranosyl) t h i o - s _ - t r i a z i n e [32]. _3 Thio-glucose (0.4 g, 1.1 x 10 mol) was d i s s o l v e d i n a c e t o n i t r i l e (6 ml) and added to a s t i r r e d a c e t o n i t r i l e s o l u t i o n (6 ml) of cyanuric -4 c h l o r i d e (0.067 g, 3.6 x 10 mol). To t h i s s t i r r e d mixture was added -3 t r i e t h y l a m i n e (0.11 g, 1.1 x 10 mol) and the mixture then allowed to s t i r at room temperature f o r 1 h. The mixture was poured i n t o i c e water (50 ml) and the p r e c i p i t a t e f i l t e r e d , washed w i t h water and d r i e d . The compound r e q u i r e d no f u r t h e r p u r i f i c a t i o n and was obtained i n 66% y i e l d , m.p. 122-124°C, [ a ] 2 2 + 9.37° (C 0.32 CHC£3). Anal, c a l c d . f o r C 4 5 H 5 7 N 3 ° 2 7 S 3 : c 46.29, H 4.88, N 3.60, S 8.24; found: C 46.10, H 4.82, 

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